JP7566331B2 - How to slow down the aging of lilies - Google Patents

Description

The present disclosure provides a transcription factor family gene (LhHFS) that controls floral senescence and a method for using the same. For example, by using techniques such as genome editing, RNA interference, or introduction of a mutagenic factor, it is possible to delay floral senescence by partially or completely eliminating the expression or function of the LhHFS gene.

No effective shelf life extenders have been developed for lilies, and no genes that control flower aging have been identified. In addition, traditional lily breeding, which is based on crossbreeding, takes about three years from crossbreeding to flowering, making it a very time-consuming and inefficient method.

Conventional lily breeding based on crossbreeding takes about three years from crossbreeding to flowering, so it takes a very long time to repeatedly crossbreed and select to impart the desired traits, which is inefficient. In addition, the shelf life of each strain has not been organized, and DNA markers related to shelf life have not been developed.

There is a demand for the development of effective quality preservation technology for flowers and ornamental plants in general. As a result of extensive research, this disclosure has identified a gene that controls the senescence of flowers in flowers and ornamental plants, which is conserved at least in all lily family plants, and has succeeded in developing a quality preservation technology.

Thus, the present disclosure provides:
(Item 1) A nucleic acid molecule comprising: (A) a nucleic acid sequence encoding the nucleic acid sequence shown in SEQ ID NO:1 or the amino acid sequence shown in SEQ ID NO:2; or (B) a sequence variant thereof,
The (B) sequence variant is
(B-1) A sequence variant having one or more substitutions, additions and/or deletions in the nucleic acid sequence of (A);
(B-2) A sequence variant having at least about 90% sequence identity to the nucleic acid sequence of (A);
(B-3) A sequence variant that hybridizes under stringent conditions to the nucleic acid sequence of (A);
(B-4) a sequence variant that is an allelic variant of (A); or (B-5) a sequence variant that is a fragment of (A) or (B-1) to (B-4);
A nucleic acid molecule, wherein the protein encoded by said sequence variant has the biological function of the protein encoded by (A).
(Item 2) The nucleic acid molecule according to any one of the above items, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO:1.
(Item 3) The nucleic acid molecule according to any one of the above items, wherein the nucleic acid molecule consists of the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.
(Item 4) (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof;
(b) a polypeptide in which one or more amino acids in the amino acid sequence shown in SEQ ID NO:2 have been mutated, the mutation including substitution, addition and/or deletion, and which has the same biological activity as the polypeptide of (a);
(c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO:1;
(d) a polypeptide encoded by a gene homologous to the gene encoding the amino acid sequence set forth in SEQ ID NO:2; or (e) a polypeptide having an amino acid sequence that is at least about 90% identical to any one of the polypeptides (a) to (d) and having biological activity similar to that of the polypeptide (a).
(Item 5) An agent that inhibits the expression of a nucleic acid sequence according to any one of the above items and/or the amount or function of said nucleic acid sequence or a protein encoded by said nucleic acid sequence, or a polypeptide according to any one of the above items.
(Item 6) The factor according to any of the above items, wherein the factor is a protein, a polypeptide, an oligopeptide, a peptide, a polynucleotide, an oligonucleotide, a nucleotide, a nucleic acid including RNA or DNA, a polysaccharide, an oligosaccharide, a lipid, a hormone, a ligand, a signal transmitter, a synthetic chemical, radiation, or a combination thereof.
(Item 7) The agent according to any one of the above items, which is selected from the group consisting of an antisense molecule, an RNAi agent, an agent for genome editing, or a mutagenesis agent.
(Item 8) A factor that inhibits LhHFS or its homologous gene.
(Item 9) The homologous gene is
(i)
A sequence variant of the nucleic acid sequence shown in SEQ ID NO:1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2, said sequence variant comprising:
(B-1) A sequence variant having one or more substitutions, additions and/or deletions in the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2;
(B-2) A sequence variant having at least about 70% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2;
(B-3) A sequence variant that hybridizes under stringent conditions to a nucleic acid sequence encoding the nucleic acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2;
(B-4) a sequence variant which is an allelic variant of the nucleic acid sequence shown in SEQ ID NO:1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2; or (B-5) a nucleic acid sequence which encodes the nucleic acid sequence shown in SEQ ID NO:1 or the amino acid sequence shown in SEQ ID NO:2, or a sequence variant which is a fragment of (B-1) to (B-4),
A sequence variant, the protein encoded by the sequence variant having the biological function of a protein encoded by the nucleic acid sequence shown in SEQ ID NO:1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2, or (ii)
(a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2 or a fragment thereof;
(b) a polypeptide in which one or more amino acids in the amino acid sequence shown in SEQ ID NO:2 have been mutated, the mutation including substitution, addition and/or deletion, and which has the same biological activity as the polypeptide of (a);
(c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO:1;
(d) a polypeptide encoded by a gene homologous to the gene encoding the amino acid sequence shown in SEQ ID NO:2; or (e) a polypeptide having an amino acid sequence that is at least about 70% identical to any one of the polypeptides (a) to (d) and having the same type of biological activity as the polypeptide (a).
(Item 10) The factor according to any of the above items, which is a protein, a polypeptide, an oligopeptide, a peptide, a polynucleotide, an oligonucleotide, a nucleotide, a nucleic acid including RNA or DNA, a polysaccharide, an oligosaccharide, a lipid, a hormone, a ligand, a signal transmitter, a synthetic chemical, or a composite molecule thereof.
(Item 11) The agent according to any one of the above items, wherein the agent is in the form of a nucleic acid.
(Item 12) The agent according to any one of the above items, which is selected from the group consisting of an antisense molecule, an RNAi agent, an agent for genome editing, or a mutagenesis agent.
(Item 13) The agent according to any one of the above items, wherein the agent is an RNAi agent for LhHFS in the form of a double-stranded nucleic acid.
(Item 14) The agent is in a double-stranded form, one of the strands being (I)
(A) a nucleic acid sequence comprising at least about 100 bases of the nucleic acid sequence shown in SEQ ID NO:1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2; or (B) a sequence variant, the other strand comprising
(II) a complementary sequence or an annealing sequence of (I);
The (B) sequence variant is
(B-1) A sequence variant having one or more substitutions, additions or deletions in the nucleic acid sequence of (A);
(B-2) A sequence variant having at least about 70% sequence identity to the nucleic acid sequence of (A);
(B-3) A sequence variant that hybridizes under stringent conditions to the nucleic acid sequence of (A);
(B-4) a sequence variant that is an allelic variant of (A); or (B-5) a sequence variant that is a fragment of (A) or (B-1) to (B-4);
The factor according to any one of the above items, wherein the protein encoded by the sequence variant has the biological function of the protein encoded by (A).
(Item 15) The factor according to any one of the preceding items, wherein (I) does not contain the nucleic acid sequence of bases 22 to 483 of SEQ ID NO:1.
(Item 16) The factor according to any one of the above items, wherein (I) comprises a nucleic acid sequence of bases 574 to 864 of SEQ ID NO:1.
(Item 17) The agent according to any one of the above items, which is a vector containing an RNAi agent for LhHFS or its homologous gene.
(Item 18) The element according to any one of the preceding items, wherein the vector comprises a promoter and a terminator.
(Item 19) The factor according to any one of the above items, wherein the factor is a factor for genome editing that suppresses the expression and/or function of LhHFS.
(Item 20) The genome editing factor according to any one of the above items, which targets a nucleic acid sequence of the following sequence in SEQ ID NO: 1.
(Item 21) The genome editing factor according to any one of the above items, which targets genome editing of the nucleic acid sequence of bases 1 to 483 of SEQ ID NO: 1.
(Item 22) A plant cell containing or modified with a factor according to any of the preceding items.
(Item 23) A plant tissue containing or modified with a factor according to any of the above items, or a plant tissue containing a cell according to any of the above items.
(Item 24) A plant organ comprising or modified by a factor according to any of the above items, or comprising a cell according to any of the above items, or a plant tissue according to any of the above items.
(Item 25) A plant or a part thereof comprising or modified by a factor according to any of the above items, or a plant or a part thereof comprising a cell according to any of the above items, a plant tissue according to any of the above items, or a plant organ according to any of the above items.
(Item 26) The plant or part thereof according to any one of the above items, wherein the plant is a plant of the lily family.
(Item 27) The plant body or part thereof according to any one of the above items, wherein the plant is a plant of the genus Lily.
(Item 28) The plant body or part thereof according to any of the above items, wherein the plant body or part thereof is a bulb, a seed, a flower, a stem, a leaf, a pollen, a scale, a grain, a cell, a tissue, a tissue culture or a corm, or a combination of all or part thereof.
(Item 29) The plant or part thereof according to any one of the preceding items, wherein the plant or part thereof includes a flower or part thereof.
(Item 30) A plant cell in which at least a portion of the LhHFS or its homologous gene contained in the plant is suppressed.
(Item 31) A plant tissue in which at least a part of LhHFS or a homologous gene thereof is suppressed, or a plant tissue containing the cell according to any one of the above items.
(Item 32) A plant organ in which at least a part of LhHFS or a homologous gene thereof contained in the plant is suppressed, or a plant organ comprising the cell according to any one of the above items or the plant tissue according to any one of the above items.
(Item 33) A plant body or part thereof in which at least a part of LhHFS or its homologous gene is suppressed, or a plant body or part thereof comprising a cell described in any of the above items, a plant tissue described in any of the above items, or a plant organ described in any of the above items.
(Item 34) The cell, tissue, organ or plant body or a part thereof according to any one of the above items, in which the expression and/or function of at least a part of the LhHFS or its homologous gene is suppressed.
(Item 35) The cell, tissue, organ, or plant body or part thereof according to any one of the above items, wherein the suppression is below the standard of reducing expression of a vacuum processing enzyme gene (VPE gene) or a cysteine protease gene (SAG12 gene) by about 50% when measured by RT-PCR.
(Item 36) The cell, tissue, organ, or plant body or part thereof according to any one of the above items, wherein the suppression is below the standard of reducing expression of a vacuum processing enzyme gene (VPE gene) by about 50% when measured by RT-PCR.
(Item 37) A cell, tissue, organ or plant body or part thereof described in any of the above items, wherein the suppression of LhHFS is achieved by a method selected from the group consisting of a reduction in gene expression level, a reduction in gene copy number, a reduction in gene amplification, a reduction in RNA activity level, a reduction in mRNA abundance, a reduction in mRNA synthesis rate, a reduction in mRNA stability, a reduction in protein activity level, a reduction in protein synthesis, a reduction in protein abundance, a reduction in protein stability, a reduction in protein enzyme activity, or a combination thereof.
(Item 38) A cell, tissue, organ or plant body or part thereof described in any of the above items, wherein at least a portion of the structural gene of LhHFS or its homologous gene has a mutation compared to the natural one, and/or the gene expression regulatory factor has a mutation compared to the natural one, and/or the cell, tissue, organ or plant body or part thereof has a non-naturally occurring factor that suppresses at least a portion of LhHFS or its homologous gene.
(Item 39) The cell, tissue, organ, or plant body, or a part thereof, according to any one of the above items, wherein the mutation or factor is provided by RNA interference or genome editing of the LhHFS gene.
(Item 40) A plant cell, plant tissue, plant organ or plant body or a part thereof, in which the gene for LhHFS or a homologous gene thereof is partially or completely mutated or deleted.
(Item 41) The plant cell, plant tissue, plant organ or plant body or a part thereof according to any one of the above items, wherein the plant is a plant of the lily family.
(Item 42) The plant cell, plant tissue, plant organ or plant body or a part thereof according to any one of the above items, wherein the plant is a plant of the genus Lily.
(Item 43) The plant cell, plant tissue, plant organ, or plant body or part thereof according to any of the above items, wherein the plant body or part thereof is a bulb, a seed, a flower, a stem, a leaf, a pollen, a scale, a grain, a cell, a tissue, a tissue culture, or a corm, or a combination of all or part thereof.
(Item 44) The plant cell, plant tissue, plant organ, or plant body or part thereof according to any one of the preceding items, wherein the plant body or part thereof includes a flower or part thereof.
(Item 45) A method for producing a plant or part thereof in which floral senescence is suppressed, the method comprising providing a state in which at least a portion of LhHFS or a homologous gene thereof contained in the plant or part thereof is suppressed.
(Item 46) The method according to any one of the above items, wherein the suppression includes suppressing the expression and/or function of at least a part of the LhHFS or its homologous gene compared to the state before the providing step.
(Item 47) The method according to any of the above items, further comprising measuring or otherwise determining that the expression and/or function of at least a portion of the LhHFS or its homologous gene is suppressed compared to the state before the providing step.
(Item 48) The method according to any of the above items, wherein the measuring or determining by other means includes measuring the abundance of mRNA of at least a portion of the LhHFS or its homologous gene, the abundance of at least a portion of the protein of the LhHFS or its homologous gene, the biological function of at least a portion of the protein of the LhHFS or its homologous gene, or a combination thereof, or calculating from the measurement results.
(Item 49) The method described in any of the above items, wherein the suppression of LhHFS or its homologous gene is achieved by a method selected from the group consisting of a reduction in gene expression level, a reduction in gene copy number, a reduction in gene amplification, a reduction in RNA activity level, a reduction in mRNA abundance, a reduction in mRNA synthesis rate, a reduction in mRNA stability, a reduction in protein activity level, a reduction in protein synthesis, a reduction in protein abundance, a reduction in protein stability, a reduction in protein enzyme activity, or a combination thereof.
(Item 50) The method according to any of the above items, wherein the suppression of the LhHFS or its homologous gene is achieved by performing RNA interference or genome editing of the LhHFS gene.
(Item 51) The method according to any one of the above items, wherein the plant is a plant of the lily family.
(Item 52) The method according to any of the preceding items, wherein the plant is a plant of the genus Lily.
(Item 53) The method according to any of the above items, wherein the plant body or part thereof is a bulb, a seed, a flower, a stem, a leaf, a pollen, a scale, a grain, a cell, a tissue, a tissue culture or a corm, or a combination of all or part thereof.
54. The method according to any of the preceding claims, wherein the plant or part thereof comprises a flower or part thereof.
(Item 55) The method comprises:
A method according to any of the above items, comprising: a step of modifying LhHFS or its homologous gene in a plant cell to introduce a mutation in part or all of it or to delete part or all of it; and a step of growing a plant cell in which LhHFS or its homologous gene has been modified to obtain a plant body or part of it.
(Item 56) The method according to any one of the above items, wherein the modification is carried out by antisense, RNA interference, mutagenesis, or genome editing.
(Item 57) A method for extending the shelf life of flowers in a plant or a part thereof, comprising:
1) providing a plant body or a part thereof comprising a factor described in any of the above items or modified by said factor, or a plant body or a part thereof comprising a cell described in any of the above items, a plant tissue described in any of the above items, or a plant organ described in any of the above items;
2) subjecting the plant or part thereof to flowering conditions;
3) subjecting the plant or a part thereof to conditions and for a period that enables the plant or a part thereof to last for a long time after flowering.
(Item 58) The method according to any one of the above items, wherein the period is longer than the period during which the shelf life of the plant body or part thereof ends in the absence of modification by the factor under the shelf life enhancing conditions.
(Item 45A) Use of a factor that suppresses LhHFS or a homologous gene thereof in a method for producing a plant body or a part thereof in which floral senescence is suppressed.
(Item 46A) The use according to any one of the above items, wherein the suppression includes suppressing the expression and/or function of at least a part of the LhHFS or its homologous gene compared to the state before the providing step.
(Item 47A) The use described in any of the above items, further comprising measuring or otherwise determining that the expression and/or function of at least a portion of the LhHFS or its homologous gene is suppressed compared to the state before the providing step.
(Item 48A) The use described in any of the above items, wherein the measuring or determining by other means includes measuring the amount of mRNA present of at least a part of the LhHFS or its homologous gene, the amount of protein present of at least a part of the LhHFS or its homologous gene, the biological function of at least a part of the protein of the LhHFS or its homologous gene, or a combination thereof, or calculating from the measurement results.
(Item 49A) The use described in any of the above items, wherein the suppression of LhHFS or its homologous gene is achieved by a method selected from the group consisting of a reduction in gene expression level, a reduction in gene copy number, a reduction in gene amplification, a reduction in RNA activity level, a reduction in mRNA abundance, a reduction in mRNA synthesis rate, a reduction in mRNA stability, a reduction in protein activity level, a reduction in protein synthesis, a reduction in protein abundance, a reduction in protein stability, a reduction in protein enzyme activity, or a combination thereof.
(Item 50A) The method according to any of the above items, wherein the suppression of the LhHFS or its homologous gene is achieved by performing RNA interference or genome editing of the LhHFS gene.
(Item 51A) The use according to any one of the above items, wherein the plant is a plant of the lily family.
(Item 52A) The use according to any one of the above items, wherein the plant is a plant of the genus Lily.
(Item 53A) The use according to any one of the above items, wherein the plant body or part thereof is a bulb, a seed, a flower, a stem, a leaf, a pollen, a scale, a grain, a cell, a tissue, a tissue culture or a corm, or a combination of all or part thereof.
(Item 54A) The use according to any one of the above items, wherein the plant body or part thereof includes a flower or part thereof.
(Item 55A) The production method includes:
A use described in any of the above items, which includes a step of modifying LhHFS or its homologous gene in a plant cell to introduce a mutation in part or all of it or to delete part or all of it; and a step of cultivating a plant cell in which LhHFS or its homologous gene has been modified to obtain a plant body or part of it.
(Item 56A) The use according to any one of the above items, wherein the modification is performed by antisense, RNA interference, mutagenesis, or genome editing.
(Item 57A) Use of an agent that suppresses LhHFS or a homologous gene thereof in a method for extending the shelf life of flowers in a plant body or a part thereof, the method comprising:
1) providing a plant body or a part thereof comprising a factor described in any of the above items or modified by said factor, or a plant body or a part thereof comprising a cell described in any of the above items, a plant tissue described in any of the above items, or a plant organ described in any of the above items;
2) subjecting the plant or part thereof to flowering conditions;
3) subjecting the plant or a part thereof to conditions and for a period of time that allows the plant or a part thereof to last after flowering.
(Item 58A) The use according to any one of the above items, wherein the period is longer than the period during which the shelf life of the plant body or part thereof ends in the absence of modification by the factor under the shelf life enhancing conditions.

These and other advantages of the present disclosure will therefore become apparent from a reading of the detailed description that follows.

The present disclosure makes it possible to delay the aging of flowers of lily family plants such as lilies and tulips by suppressing the expression of a transcription factor family gene (LhHFS), thereby extending the period during which the flowers can be enjoyed.

FIG. 1 shows photographs of the state of flowers after flowering in wild-type (Cont) and LhHFS expression suppression line (LNR18-2). The number of days after flowering is indicated at the top of the photographs. The photographs in the upper row show wild-type lilies from 0 to 7 days after flowering, and the photographs in the lower row show lilies of the LhHFS expression suppression line from 0 to 9 days after flowering. Browning of the perianth was observed 5 days after flowering in the wild-type, and 8 days after flowering in the LhHFS expression suppression line. The arrows indicate that the shelf life ended 4 days after flowering in the wild-type, and 7 days after flowering in the LhHFS expression suppression line. 2 shows the relative expression level of the LhHFS gene in perianths 4 days after flowering in the wild type (Cont) and the LhHFS expression-suppressed line (LNR18). The vertical axis of the graph shows the relative expression level of the LhHFS gene, and the axes correspond, from left to right, to the wild type (Cont) and the LhHFS expression-suppressed lines (LNR18-2 and LNR18-15).

The present disclosure will be described below. Throughout this specification, singular expressions should be understood to include the concept of the plural form, unless otherwise specified. Therefore, singular articles (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will take precedence.

(Definition)
In this specification, the term "about" means ±10% of the value that follows. It should be understood that in this specification, even if the term "about" is not explicitly stated, no waiver of the numerical range in which the term "about" is not stated is intended.

As used herein, "LhHFS" refers to a gene having the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, a protein having the amino acid sequence set forth in SEQ ID NO:2, a biologically functional derivative thereof, or a biologically functional fragment thereof, or a homolog thereof, or a variant encoded by a nucleic acid that hybridizes to a nucleic acid encoding this protein under high or low stringency conditions, as found in this disclosure. Those skilled in the art will understand whether only the nucleic acid molecule or the protein, or both, are meant, as appropriate, depending on the context. "Mutants" or "variants" also include, but are not limited to, molecules that contain a region that is substantially homologous to the original protein, which in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical over the same size amino acid sequence or when compared to aligned sequences as determined by computer homology programs known in the art, such as LALIGN (https://embnet.vital-it.ch/software/LALIGN_form.html), or nucleic acids encoding such molecules are capable of hybridizing to sequences encoding the original protein under stringent, moderately stringent or non-stringent conditions. This refers to proteins that are the product of modifications of naturally occurring proteins by amino acid substitutions, deletions, and additions, respectively, such that the derivatives still exhibit the biological functions of the naturally occurring proteins, although not necessarily to the same degree. The biological functions of such proteins can be examined, for example, by suitable available in vitro assays described herein or known in the art.

In this disclosure, LhHFS is primarily discussed in lilies, but it is known that many plants other than lilies, such as tulips (SEQ ID NO: 4), express genes homologous to the LhHFS gene. In particular, the tulip homologous gene has approximately 71% sequence identity to lily LhHFS, and based on this interspecies sequence conservation, it is understood that these plants, particularly liliaceae plants, are also within the scope of this disclosure.

As used herein, "biologically functional" refers to a polypeptide, i.e., a fragment or derivative, that has a structural, regulatory, or biochemical function of a protein, such as biological activity, according to the embodiment to which the polypeptide, i.e., fragment or derivative, of the present disclosure relates.

In the context of this disclosure, "(gene or protein) suppressing factor" refers to a factor (e.g., a molecule or substance) that acts directly or indirectly on a gene of interest or its homologous gene, or on a protein encoded by these genes, and can temporarily or permanently suppress, reduce or eliminate the function of the gene of interest or its homologous gene, or the protein encoded by these genes. The suppressing factor may be an inhibitor (substance), examples of which include antibodies or antigenic fragments thereof, antisense oligonucleotides, RNAi factors such as siRNA, factors for genome editing, mutagenic factors, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes and peptidomimetics, and the like, including binding proteins or binding peptides directed against the gene of interest or its homologous gene, or against the protein encoded by these genes, particularly against the active site of the protein encoded by the gene of interest or its homologous gene, as well as nucleic acids directed against the gene of interest or its homologous gene. Nucleic acids for a gene of interest or its homologous genes refer to double-stranded or single-stranded DNA or RNA, or modifications or derivatives thereof, that inhibit, for example, the expression or activity of the gene of interest or its homologous genes, or the proteins encoded by these genes, and include, without limitation, antisense nucleic acids, aptamers, siRNAs (small interfering RNAs) and ribozymes. Nucleic acids for a gene of interest or its homologous genes also include factors for genome editing that inhibit, for example, the expression or activity of the LhHFS gene or its homologous genes, and factors for genome editing include, for example, artificial restriction enzymes (artificial nucleases) and RNA-guided nucleases. In this specification, the term "binding protein" or "binding peptide" for a protein encoded by a gene of interest or its homologous genes refers to a type of protein or peptide that binds to the protein encoded by the gene of interest or its homologous genes, and includes, but is not limited to, polyclonal or monoclonal antibodies, antibody fragments, and protein scaffolds directed against the protein encoded by the gene of interest or its homologous genes.

In this specification, the term "signal transmitter" refers to a substance that transmits information to regulate physiological activity or biological functions in the body. Representative examples of "signal transmitters" include plant hormones.

In this specification, the term "antisense molecule" refers to a molecule that can specifically suppress or reduce the expression of a target gene. More specifically, it refers to a molecule that can reduce the amount of protein expression by specifically reducing the amount of mRNA of a gene that has a nucleotide sequence region complementary to a certain nucleotide sequence introduced into a cell. Antisense techniques are broadly divided into a method of directly introducing an RNA molecule complementary to the mRNA produced from the target gene into a cell, and a method of introducing a construction vector that can express RNA complementary to the target gene into a cell, but the latter is more common in plants. Specifically, a DNA sequence complementary to the nucleotide sequence of the target gene is linked to an appropriate promoter, and an expression vector that expresses artificial mRNA under its control is constructed and introduced into a cell, as well as a method using an expression vector designed to form double-stranded RNA in a cell. The basic structure of an antisense molecule is created by linking one type of DNA sequence complementary to a certain target gene under a promoter, and then linking another identical one in the opposite direction. In the single-stranded mRNA transcribed from this construct gene, one type of nucleotide sequence portion linked in the opposite direction is complementary, so they pair up to form a double-stranded RNA state with a hairpin-like secondary structure, which triggers the degradation of the mRNA of the target gene according to the RNAi mechanism.

As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, which is generally known in the art and is a biological process mediated by RNAi factors that inhibit or downregulate gene expression in cells. For example, it refers to the phenomenon in which homologous mRNA is specifically degraded and synthesis of gene products is suppressed by introducing factors that cause RNAi, such as double-stranded RNA (also called dsRNA), into cells, and the technology used therefor. For RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al. , 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbasiretal. , 2001, Nature, 411, 494-498; and Kreutzer et al., WO 00/44895; Zernicka-Goetz et al., WO 01/36646; Fire, WO 99/32619; Plaetinck et al., WO 00/01846; Mello and Fire, WO 01/29058; Deschamps-Depaillette, WO 99/07409 and Li et al., WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al. , 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al. , 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al. , 2002, RNA, 8, 842-850; Reinhart et al. , 2002, gene & Dev. , 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831.) As used herein, the term RNAi is also understood to be synonymous with other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, epigenetics, and the like.

As used herein, "RNAi factors" include nucleic acid molecules or analogs thereof that inhibit gene expression, such as RNA-based molecules. RNAi factors may be factors that destroy or cleave specific mRNAs, or may be factors that block the processing or translation of RNA, such as mRNA. RNAi factors include, but are not limited to, silencing RNA (siRNA), endoribonuclease-prepared siRNA (esiRNA), short hairpin RNA (shRNA), or microRNA (miRNA), and short interfering nucleic acid (siNA). These factors refer to any nucleic acid molecule that can inhibit or downregulate gene expression or viral replication by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. These terms may also refer to individual nucleic acid molecules, a plurality of such nucleic acid molecules, or a pool of such nucleic acid molecules. These molecules may be double-stranded nucleic acid molecules that include self-complementary sense and antisense regions. Here, the antisense region includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and a sense region that has a nucleotide sequence or a portion thereof that corresponds to the target nucleic acid sequence. These molecules can be assembled from two separate oligonucleotides, one of which is the sense strand and the other the antisense strand, where the antisense and sense strands are self-complementary (i.e., each strand contains a nucleotide sequence that is complementary to a nucleotide sequence in the other strand, such that the antisense and sense strands form a double-stranded or duplex structure, where, for example, the double-stranded region can be about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, but can be longer. The antisense strand contains a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand contains a nucleotide sequence that corresponds to a target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, these molecules can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of these molecules are complementary to each other, where, for example, the double-stranded region can be about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, but can be longer. The antisense region is linked by a nucleic acid linker or a non-nucleic acid linker. These molecules can be polynucleotides having a double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structure, including self-complementary sense and antisense regions. Here, the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a separate target nucleic acid molecule, and a sense region having a nucleotide sequence or a portion thereof corresponding to the target nucleic acid sequence. These molecules can be circular single-stranded polynucleotides having two or more loop structures and a stem that comprises the self-complementary sense and antisense regions. Here, the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and a sense region having a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule, and a nucleotide sequence that is corresponding to the target nucleic acid sequence or a portion thereof, and the circular polynucleotide is an in They may be processed in vivo or in vitro to generate active molecules capable of mediating RNAi. These agents may also include single-stranded polynucleotides having a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule (e.g., these agents do not require that a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof be present within the agent). Single-stranded polynucleotides may further include a terminal phosphate group, such as a 5' phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), a 5',3'-diphosphate, or the like. In certain embodiments, the agents that inhibit LhHFS of the present disclosure include separate sense and antisense sequences or regions. Here, the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules known in the art, or alternately non-covalently linked by ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions and/or stacking interactions. In some embodiments, the LhHFS inhibitor of the present disclosure comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In another embodiment, the LhHFS inhibitor of the present disclosure interacts with the nucleotide sequence of the target gene so as to inhibit expression of the target gene. As used herein, the LhHFS inhibitor is not necessarily limited to molecules that contain only RNA, but also encompasses chemically modified nucleotides and non-nucleotides. In some embodiments, when the present disclosure is a small interfering nucleic acid molecule, it may lack 2' hydroxy (2'-OH)-containing nucleotides. In some embodiments, the present disclosure may be a small interfering nucleic acid that does not require the presence of a nucleotide with a 2' hydroxyl group to mediate RNAi. Thus, when the present disclosure is a small interfering nucleic acid molecule, it may not contain ribonucleotides (e.g., nucleotides with a 2'-OH group). However, if the presence of ribonucleotides in the LhHFS inhibitor is not required to maintain RNAi, it may have an attached linker or other attached or associated group, moiety, or strand that includes one or more nucleotides with a 2'-OH group. In some cases, the LhHFS inhibitor of the present disclosure may include ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The LhHFS inhibitor herein may be a nucleic acid molecule capable of mediating sequence-specific RNAi, such as small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), small interfering oligonucleotide, small interfering nucleic acid, small interfering modified oligonucleotide, chemically modified siRNA, or post-transcriptional gene silencing RNA (ptgsRNA).

As used herein, a "factor for genome editing" refers to a factor that causes genome editing in a cell, tissue, organ, or organism into which it is introduced. A "factor for genome editing" may be any factor that causes genome editing by a method known in the art, i.e., CRISPR/Cas9, TALEN, ZFN, meganuclease, or the like. Factors for genome editing include, but are not limited to, artificial restriction enzymes (artificial nucleases), RNA-guided nucleases, guide RNAs (gRNAs), and the like.

As used herein, the term "mutagenist" refers to a factor that promotes the induction of mutations in a target gene. "Mutagenists" include chemical substances such as ethyl methanesulfonate (EMS), as well as energy such as light, radiation, heat, and electricity.

As used herein, "homologous genes" refer to genes with high homology between two or more sequences. The "homology" of a gene refers to the degree of identity between two or more gene sequences, and generally, "homology" refers to a high degree of identity or similarity. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes have homology can be determined by direct comparison of the sequences or, in the case of nucleic acids, by hybridization methods under stringent conditions. When directly comparing two gene sequences, the genes have homology if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical. Thus, as used herein, "homolog" or "homologous gene product" refers to a protein in another species, preferably a plant, that exerts the same biological function as a protein component of a complex as further described herein. Such homologs may also be referred to as "orthologous gene products." Algorithms for detecting orthologous gene pairs from a particular plant or other species use the entire genome or partial sequences of these organisms. When analyzing the homology of partial sequences, the LALIGN program (https://embnet.vital-it.ch/software/LALIGN_form.html) is typically used in the present disclosure. When analyzing with the LALIGN program, a person skilled in the art can compare sequence identity according to the instructions on the website. Details of the analysis method are provided in X. Huang and W. Miller, Adv. Appl. Math. (1991) 12:337-357, etc. Based on sequence homology of the genes encoding the proteins provided herein to genes of other species, it is also possible to isolate homologues of the proteins described herein by applying conventional techniques to clone the respective genes and express the proteins from such genes, or by isolating the proteins of other species by isolating similar complexes according to the methods provided herein or other suitable methods well known in the art.

In this specification, the numerical value of identity, etc., "70% or more" may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%, or may be within the range of any two of the starting numerical values. The above-mentioned "identity" is calculated by calculating the ratio of the number of homologous amino acids in two or more amino acid sequences according to the known method as described above. Specifically, before calculating the ratio, the amino acid sequences of the amino acid sequence group to be compared are aligned, and gaps are introduced into a part of the amino acid sequence if necessary to maximize the ratio of identical amino acids. Methods for alignment, calculation of the ratio, comparison, and computer programs related thereto are conventionally well known in the art (for example, BLAST, etc., as described above). In this specification, "identity" and "similarity" can be expressed as values measured by BLAST of NCBI, unless otherwise specified. When comparing amino acid sequences with BLAST, the algorithm Blastp can be used with default settings. Measurement results are quantified as Positives or Identities.

As used herein, when referring to genes, a "vector" refers to a vector capable of transferring a polynucleotide sequence of interest into a cell of interest. Examples of such vectors include those capable of autonomous replication in host cells, such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals, and individual plants, or those capable of integration into a chromosome, and which contain a promoter in a position suitable for transcription of the polynucleotide of the present disclosure. As used herein, a vector may be an expression vector, a recombinant vector, or the like.

As used herein, the term "promoter" refers to a region on DNA that determines the start site of gene transcription and directly regulates its frequency, and is a base sequence to which RNA polymerase binds to initiate transcription. The promoter region can usually be estimated by predicting the protein-coding region in the genome sequence using DNA analysis software. The estimated promoter region varies for each structural gene, but is usually located upstream of the structural gene, but is not limited to this, and may also be located within or downstream of the structural gene. In the present disclosure, any promoter that exerts the function of a factor that causes RNA interference of LhHFS may be used.

As used herein, a "terminator" refers to a sequence that is located downstream of a protein-coding region of a gene and is involved in terminating transcription and adding a polyA sequence when DNA is transcribed into mRNA. Terminators include, but are not limited to, the CaMV35S terminator, the nopaline synthase gene terminator (Tnos), and the tobacco PR1a gene terminator. In the present disclosure, any sequence that exhibits terminator activity in plants can be used.

In this specification, the "expression" of genes, polynucleotides, polypeptides, proteins, etc. means that the genes, etc. are subjected to a certain action in vivo to become a different form. Preferably, it means that the genes, polynucleotides, etc. are transcribed and translated to become a polypeptide, but transcription to produce mRNA is also one form of expression. For example, the expression level of LhHFS can be determined by any method. Specifically, the expression level of LhHFS can be known by evaluating the amount of LhHFS mRNA, the amount of LhHFS protein, and the biological function of LhHFS protein. The amount of LhHFS mRNA and protein can be determined by methods detailed elsewhere in this specification or other methods known in the art.

As used herein, "inhibition of the function" of a gene, polynucleotide, polypeptide, protein, etc. refers to inhibition of the biological activity of the gene, etc. The "inhibition of the function" of LhHFS can be confirmed by, for example, measuring the expression of the vacuum processing enzyme gene (VPE gene) or the cysteine protease gene (SAG12 gene) using the RT-PCR method. The inhibition of the function of LhHFS can be confirmed, for example, by a reduction in the expression of the VPE gene or the SAG12 gene by about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%, preferably about 50%, compared to the expression in a control, when measured by the RT-PCR method.

In this specification, "plant" is a general term for organisms belonging to the kingdom Plantae, and is characterized by the presence of chlorophyll, hard cell walls, abundant and persistent embryonic tissue, and the inability to move. Typically, plants refer to flowering plants that form cell walls and have the ability to assimilate chlorophyll. "Plants" include both monocotyledonous and dicotyledonous plants. Preferred plants include, for example, plants of the "Liliaceae" family. Representative plants of the "Liliaceae" family include lilies, tulips, dogtooth violets, and Japanese black lilies.

In this specification, the term "tissue" of an organism refers to a group of cells that have a certain similar function within that group. Thus, a tissue may be part of an organ. Organs often contain cells that have the same function, but there may also be a mixture of cells with slightly different functions. Therefore, in this specification, tissue may contain a mixture of various cells as long as they share certain characteristics. In addition to those that make up a differentiated plant body, "tissue" also includes plant cell masses such as "callus."

In this specification, the term "organ" refers to a structure that has an independent form and is formed by combining one or more types of tissue to perform a specific function. In plants, examples include, but are not limited to, roots, bulbs, stems, trunks, leaves, flowers, seeds, scales, kernels, germs, embryos, fruits, endosperms, and cornels.

As used herein, the term "organism" (or "plant" in the case of a plant) is used in the broadest sense in the art to refer to an entity (or plant) that engages in life phenomena, and typically has various characteristics such as cell structure, proliferation (self-reproduction), growth, regulation, metabolism, and repair ability, and typically has as its basic attributes proliferation involving genetics governed by nucleic acids and metabolism governed by proteins. Organisms include prokaryotes and eukaryotes (plants, animals, etc.). Preferably, in the present disclosure, an organism may be a plant. As used herein, preferably, such a plant may be fertile. More preferably, such a plant may produce seeds. It is contemplated that the genetic constructs, agents, compositions, and methods of the present disclosure function not only in liliaceae plants, but also in plants in general.

In this specification, the term "lily family" plants is intended to include the original species and hybrids of plants belonging to the biological taxonomy family Liliaceae. Examples of plants in the lily family include the genus Lilium, Tulipa, Erythronium, Fritillaria, Amana, Bowiea, Calochortus, Cardiocrinum, Clint onia), Gagea, Lloydia, Medeola, Nomocharis, Notholirion, Prosartes, Streptopus, and Tricyrtis are some of the genera.

In this specification, the term "Lilium" plants is intended to include the original species and hybrids of plants belonging to the genus Lilium in the family Liliaceae in biological taxonomy. Thus, the "Lilium" plants include original species such as trumpet lilies, kanoko lilies, escaping lilies, tiger lilies, mountain lilies, and bamboo lilies, as well as hybrids such as oriental hybrids, asiatic hybrids, longiflorum hybrids, martagon hybrids, and trumpet hybrids. In the examples, the aging retardation effect is demonstrated using oriental hybrid varieties, but since the homology of the LhHFS gene is very high between hybrids, it is believed that the aging retardation effect will also be demonstrated in hybrids of lilies other than oriental hybrids. Examples of "oriental hybrids" include varieties such as tiara, casablanca, and acapulco.

In this specification, the term "bulb" refers to a storage organ that is formed when nutrients accumulate in a specific part of a plant, such as the root, stem, or leaves, causing the part to deform and swell. Representative plants that produce bulbs include lilies, tulips, and hyacinths.

As used herein, "seed" or "seed" refers to the reproductive organ of a seed plant.

In this specification, the term "flower" refers to a collection of organs for plant reproduction. The term "flower" refers to a combination of the stalk, perianth, stamens, pistil, etc. In this specification, the term "flower" may be provided as a "cut flower" that is separated from the underground part, or as a "potted flower" or a flower in a flower bed that retains the underground part. The term "flower" may generally be provided as an object of ornamental appreciation. The term "cut flower" refers to a flower alone, or a combination of tissues and/or organs that include a flower. For example, the term "cut flower" may be a combination of flowers, stems, and/or leaves. Specifically, the term "cut flower" may be a combination of flowers, stems, and leaves. The term "potted flower" refers to a plant that is cultivated in a flowerpot and blooms. The term "part of a flower" refers to a flower that is missing at least a part of the elements that make up the above-mentioned "flower."

In this specification, "perianth" refers to the entire name combining "corolla" and "sepals." "Corolla" refers to an organ consisting of multiple petals.

In this specification, "receptacle" refers to the part at the tip of the stem where the perianth, corolla, petals, stamens, pistil, and sepals are attached.

As used herein, "stem" refers to the organ in a plant that supports flowers and leaves.

As used herein, "leaf" refers to the organ in a plant that performs photosynthesis and respiration.

As used herein, "pollen" refers to the cells produced by the anthers present in the stamens of flowers.

In this specification, "scale" or "scale leaf" refers to a modified leaf that protects the buds and bulbs of a plant body.

In this specification, "corn" refers to the small bulbs that form around the bulb.

In this specification, "bulbul" refers to an organ in a plant body that is formed when a lateral bud stores nutrients and enlarges.

As used herein, "callus" refers to a mass of undifferentiated plant cells.

As used herein, "senescence" refers to the phenomenon or process of a decline in the function of an organism, cell, tissue, or organ, or the function that integrates them, that occurs after maturity and before the death of the organism. When referring to "flower senescence" in this disclosure, it particularly refers to the phenomenon (or the time course itself) that occurs over time from after flowering (or sometimes before flowering) to the end of the shelf life (loss of ornamental value).

In this specification, "longevity" and "flower senescence" are defined from a horticultural perspective, and are evaluated by the "browning" of the entire flower, or the organs or tissues that compose the flower (for example, petals or similar organs). When the b* value in the L*a*b* color system using a colorimeter increases to a certain degree compared to the perianth immediately after blooming (in a particularly strict evaluation, when the b* value in the L*a*b* color system using a colorimeter increases by 10 or more compared to the perianth immediately after blooming), the flower is evaluated as having "browning" and is evaluated as "senescence" in this disclosure, and the state before being evaluated as "senescence" is expressed as "longevity". In addition, "inhibition of flower senescence" is defined from a horticultural perspective, and in this specification, when the increase in the b* value during flower senescence is significantly inhibited, it is referred to as inhibition of flower senescence. Inhibition may be in the degree of increase, or may be evaluated by the extension of the time until a certain degree of increase. Shelf life and aging can also be evaluated using other methods (e.g., state of wilting, presence or absence of detachment).

Without wishing to be bound by theory, aging is a phenomenon or process in which cells die spontaneously, and is considered to be a type of programmed cell death. An autophagy-like phenomenon has long been observed in plants such as morning glories during the aging of flower cells.

"Floral senescence" or its inhibition can be evaluated by observing the time from when the whole flower, or an organ or tissue that constitutes the flower (e.g., perianth, corolla, petals, or similar organs) opens until it turns brown (or by other equivalent evaluation methods). In this specification, floral senescence is typically evaluated using a lily family plant, but the present disclosure is not limited thereto.

In this specification, "flowering conditions (or period)" refers to the conditions (or period) under which flowering plants normally grow.

In this specification, "long-lasting conditions (or period)" refers to the conditions (or period) during which a flowering plant will remain in bloom.

As used herein, "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term may also include those assembled into complexes of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety). The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art.

As used herein, "amino acids" may be either natural or unnatural, so long as they satisfy the objectives of this disclosure.

As used herein, "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. The terms also include "oligonucleotide derivatives" or "polynucleotide derivatives." "Oligonucleotide derivatives" or "polynucleotide derivatives" refer to oligonucleotides or polynucleotides that contain derivatives of nucleotides or have unusual bonds between nucleotides, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which a phosphodiester bond in an oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which a phosphodiester bond in an oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which a ribose and a phosphodiester bond in an oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which uracil in an oligonucleotide has been replaced with C-5 propynyl uracil, oligonucleotide derivatives in which uracil in an oligonucleotide has been replaced with C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in an oligonucleotide has been replaced with C-5 propynyl cytosine, oligonucleotide derivatives in which cytosine in an oligonucleotide has been replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA has been replaced with 2'-O-propyl ribose, and oligonucleotide derivatives in which ribose in an oligonucleotide has been replaced with 2'-methoxyethoxy ribose. Unless otherwise indicated, a particular nucleic acid sequence is also intended to encompass its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotides" may be natural or non-natural.

As used herein, "gene" refers to a factor that determines a genetic trait. It is usually arranged in a specific order on a chromosome. A gene that determines the primary structure of a protein is called a structural gene, and a gene that controls its expression is called a regulatory gene. As used herein, "gene" can refer to a "polynucleotide," "oligonucleotide," and "nucleic acid."

As used herein, "genome" refers to the entirety of an organism's genetic information. A genome can be encoded in either DNA or RNA. A genome can include coding regions that code for proteins as well as non-coding regions.

Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may be referred to by their commonly accepted one-letter codes. In this specification, comparisons of amino acid and base sequence similarity, identity, and homology are calculated using BLAST, a sequence analysis tool, with default parameters. Identity searches can be performed, for example, using BLAST 2.10.1 (published 2020.6.18) from NCBI. The identity value in this specification usually refers to the value when aligned under default conditions using the above-mentioned BLAST. However, if a higher value is obtained by changing the parameters, the highest value is taken as the identity value. If identity is evaluated in multiple regions, the highest value among them is taken as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.

In this specification, "polynucleotides that hybridize under stringent conditions" refers to well-known conditions commonly used in the art. Such polynucleotides can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like, with a polynucleotide selected from the polynucleotides disclosed herein as a probe. Specifically, the polynucleotide refers to a polynucleotide that can be identified by hybridizing a filter on which DNA derived from a colony or plaque is immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using a 0.1 to 2 times concentration of SSC (saline-sodium citrate) solution (the composition of a 1 times concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). Hybridization is described in Molecular Cloning 2nd ed. This can be carried out according to the methods described in experimental textbooks such as "Current Protocols in Molecular Biology, Supplement 1-38" and "DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995)". Here, sequences that hybridize under stringent conditions preferably exclude sequences that contain only A or only T sequences. Thus, the polypeptides used in the present disclosure (e.g., proteins encoded by LhHFS) also include polypeptides encoded by nucleic acid molecules that hybridize under stringent conditions to nucleic acid molecules that encode the polypeptides specifically described in the present disclosure. These low stringency conditions include hybridization at 40°C for 18-20 hours in a buffer containing 35% formamide, 5xSSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% BSA, 100μg/ml denatured salmon sperm DNA, and 10% (weight/volume) dextran sulfate, washing at 55°C for 1-5 hours in a buffer consisting of 2xSSC, 25mM Tris-HCl (pH 7.4), 5mM EDTA, and 0.1% SDS, and washing at 60°C for 1.5 hours in a buffer consisting of 2xSSC, 25mM Tris-HCl (pH 7.4), 5mM EDTA, and 0.1% SDS.

The functional equivalents of LhHFS used in this disclosure can be found by searching databases, etc. As used herein, "searching" refers to the use of a nucleic acid sequence, electronically or by biological or other methods, to find other nucleic acid sequences having particular functions and/or properties. Examples of electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85:2444-2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147:195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)). Biological searches include, but are not limited to, stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays in which genomic DNA is attached to a glass plate (microarray assay), PCR, and in situ hybridization. In this specification, the gene used in this disclosure is intended to include corresponding genes identified by such electronic searches and biological searches.

As the functional equivalent of the present disclosure, an amino acid sequence in which one or more amino acids have been inserted, substituted, or deleted, or which has been added to one or both termini, can be used. In this specification, "an amino acid sequence in which one or more amino acids have been inserted, substituted, or deleted, or which has been added to one or both termini" means that the amino acid sequence has been modified by a well-known technical method such as site-directed mutagenesis, or by natural mutation, by substitution of a number of amino acids to the extent that may occur naturally.

As used herein, an "isolated" biological factor (e.g., a gene (LhHFS), a nucleic acid, or a protein, etc.) refers to a substance or biological factor from which at least some of the factors naturally associated with the substance or biological factor have been removed, and is used to distinguish the substance or biological factor from a natural product. Thus, the purity of the biological factor in an isolated biological factor is usually higher (i.e., it is concentrated) than the state in which the biological factor normally exists.

As used herein, a "purified" substance or biological agent (e.g., a nucleic acid or protein, etc.) refers to a biological agent from which at least a portion of the factors naturally associated with the biological agent have been removed. Thus, typically, the purity of the biological agent in a purified biological agent is greater (i.e., concentrated) than the state in which the biological agent is normally present. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological agent is present. The substances used in this disclosure are preferably "purified" substances.

As used herein, a "corresponding" amino acid or nucleic acid refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule that has or is predicted to have the same action as a specific amino acid or nucleotide in a polypeptide or polynucleotide that is used as a reference for comparison. For example, in an enzyme molecule, it refers to an amino acid that is present at a similar position in the active site and contributes to catalytic activity in a similar manner. For example, in the case of an antisense molecule, it may be a similar portion in an orthologue that corresponds to a specific portion of the antisense molecule. The corresponding amino acid may be, for example, a specific amino acid that is cysteinized, glutathionylated, S-S bond formed, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, or the like. Alternatively, the corresponding amino acid may be an amino acid that is responsible for dimerization. Such a "corresponding" amino acid or nucleic acid may be a region or domain that spans a certain range. In such a case, it is referred to as a "corresponding" region or domain in this specification.

As used herein, a "corresponding" gene (e.g., a polynucleotide sequence or molecule) refers to a gene (e.g., a polynucleotide sequence or molecule) that has or is expected to have the same function in a given species as a given gene in a species that is the basis for comparison, and when there are multiple genes with such function, it refers to those that have the same evolutionary origin. Thus, a gene that corresponds to a given gene may be an ortholog of that gene. Thus, for example, LhHFS in lilies can be found in tulips. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in a given plant (e.g., lily) can be found by searching a sequence database for that plant (e.g., tulip) using a sequence such as SEQ ID NO: 1 or SEQ ID NO: 3 as a query sequence, and a reference gene for the corresponding gene (e.g., LhHFS, etc.).

As used herein, a "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of the fragment can be changed as appropriate depending on the purpose. For example, the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In addition, for a polynucleotide, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In the present specification, such fragments are understood to be within the scope of the present disclosure, for example, if the full-length fragment functions as a marker, as long as the fragment itself also functions as a marker.

As used herein, "activity" refers to the function of a molecule in the broadest sense. Activity generally includes, but is not limited to, the biological, biochemical, physical, or chemical functions of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules, stability, and the ability to localize to a specific subcellular location. Where applicable, the term also relates to the function of a protein complex in the broadest sense. In the present disclosure, the "activity" of LhHFS is typically transcription activity (also referred to herein as "LhHFS transcription activity") or gene expression induction activity, and more specifically may be senescence-related gene induction activity or programmed cell death-related gene induction activity.

In this specification, the term "biological function" refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in a living body, and includes, but is not limited to, the production of specific antibodies, enzyme activity, and the conferring of resistance. In the present disclosure, for example, the function of LhHFS, etc. to promote or control flower senescence, etc., is included, but is not limited to these. In this specification, a biological function may be exerted by "biological activity". In this specification, "biological activity" refers to the activity that a certain factor (e.g., polynucleotide, protein, etc.) may have in a living body, and includes activities that exert various functions (e.g., transcription activity or gene expression induction activity), and also includes, for example, the activity of activating or inactivating another molecule by interacting with a certain molecule. When two factors interact with each other, the biological activity is determined by the binding between the two molecules and the biological change that occurs as a result of this. For example, when one molecule is precipitated using an antibody and the other molecule is also co-precipitated, the two molecules are considered to be bound. Therefore, observing such co-precipitation is one method of determination. In the case of the activity of delaying flower senescence, as disclosed herein, such activity is included. For example, if a factor is an enzyme, its biological activity includes its enzymatic activity. In another example, if a factor is a ligand, its biological activity includes the binding of the ligand to a corresponding receptor. Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus), such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the present disclosure, or, for example, a measure of the amount of an upstream or downstream protein or other similar function after some stimulus or event.

In this specification, the "expression" of a gene, polynucleotide, polypeptide, etc. means that the gene, etc. is subjected to a certain action in vivo and becomes a different form. Preferably, it means that the gene, polynucleotide, etc. is transcribed and translated into the form of a polypeptide, but transcription to produce mRNA can also be one form of expression. More preferably, the form of such a polypeptide can be one that has been subjected to post-translational processing (derivatives as referred to in this specification). For example, the expression level of LhHFS can be determined by any method. Specifically, the expression level of LhHFS can be known by evaluating the amount of LhHFS mRNA, the amount of LhHFS protein, and the biological activity of LhHFS protein. The amount of LhHFS mRNA and protein can be determined by the method described in this specification.

In this specification, the term "functional equivalent" refers to any entity that has the same intended function but a different structure compared to the original entity of interest. Therefore, when referring to "LhHFS or its functional equivalent" or "the group consisting of LhHFS and its functional equivalents," it is understood that the term includes not only LhHFS itself, but also mutants or variants of LhHFS (e.g., amino acid sequence variants, etc.) that have the effect of delaying flower senescence, as well as LhHFS itself or those that can be changed into this mutant or variant of LhHFS at the time of acting (e.g., nucleic acid encoding LhHFS itself or a mutant or variant of LhHFS, and vectors, cells, etc. containing the nucleic acid). In this disclosure, it is understood that the functional equivalent of LhHFS can be used in the same way as LhHFS, even if not specifically mentioned.

The modified amino acid sequence of LhHFS may be, for example, one in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted, or deleted, or added to one or both ends. The modified amino acid sequence may preferably be an amino acid sequence having one or more (preferably one or several, or one, two, three, or four) conservative substitutions in the amino acid sequence of LhHFS. Here, "conservative substitution" means replacing one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, a hydrophobic residue may be replaced with another hydrophobic residue, a polar residue may be replaced with another polar residue having the same charge, and the like. Functionally similar amino acids that can be replaced in this way are known in the art for each amino acid. Specific examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In this specification, the terms "drug", "agent" or "factor" (all of which correspond to the English term agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radiation, heat, electricity, etc.) as long as it can achieve the intended purpose. Such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof. Factors specific to a polynucleotide typically include, but are not limited to, polynucleotides that have a certain sequence homology (e.g., 70% or more sequence identity) with respect to the sequence of the polynucleotide and polypeptides such as transcription factors that bind to promoter regions. Representative factors specific to a polypeptide include, but are not limited to, an antibody or a derivative or analog thereof (e.g., a single-chain antibody) specifically directed against the polypeptide, a specific ligand or receptor when the polypeptide is a receptor or ligand, and a substrate when the polypeptide is an enzyme.

(Genetic engineering, gene modification technology)
In the present disclosure, examples of genetic engineering and gene modification techniques used to suppress LhHFS or its homologous genes include genome editing, antisense, RNAi, and mutagenesis.

As used herein, "genome editing" refers to a technique for introducing mutations to specifically alter (e.g., suppress, reduce, improve or increase) the expression of a target gene. More specifically, it is a technique that uses a site-specific nuclease to cleave at a target position in the genomic sequence of a target gene, thereby generating a single-stranded or double-stranded break at a specific position in the genome. The break in the genome thus generated is usually repaired by processes such as homologous recombination (HDR) and non-homologous end repair (NHEJ). Random deletion or insertion of several bases in the target gene causes genetic mutations such as nonsense mutations, missense mutations, and/or frameshifts, and normal (biologically functional) proteins are not produced. Genome editing techniques include CRISPR/Cas9, TALEN, ZFN, meganuclease, and other techniques. General techniques for genome editing are described, for example, in Cox et al., Nat. Med. 21: 121-131 (2015); Zhang et al., Genome Biol. 19: 210 (2018). Introduction of mutations into target genes using genome editing is advantageous in that it is possible to specifically introduce mutations into specific genes to produce varieties with desired properties more efficiently than conventional methods such as mutagenesis or repeated crossbreeding. In addition, under the system in Japan at the time of filing this application, plant cells, plant tissues, plant organs, or plant bodies or parts thereof that have been produced using "genome editing" technology without residual foreign genes can be used without undergoing safety screening under the Cartagena Act.

When introducing a mutation into a gene by genome editing, methods using CRISPR/Cas9, a complex in which the part involved in binding to the target DNA is RNA and the part involved in DNA cleavage is protein, and TALEN, ZFN, etc., in which both the part involved in binding to the target DNA and the part involved in DNA cleavage are proteins, can be mentioned. For example, in the method using CRISPR/Cas9, guide RNA, Cas9, etc. are introduced into the target cell, and in the method using TALEN or ZFN, a fusion protein in which a DNA binding domain and a nuclease are fused, etc. are introduced into the target cell, thereby introducing the mutation into the genome. Methods for introducing into the target cell include the agrobacteria method, the RNA virus vector method, the plasma treatment method, the particle gun (bombardment) method, the PEG method, the electroporation method, etc.

A suitable method for introducing a mutation into a genome by genome editing may be a method in which a target base sequence is cleaved and then a mutation occurs when the cleavage site is naturally repaired, or a method in which a DNA fragment having one or several mutated bases in a sequence homologous to the target base sequence is introduced into a cell, the target base sequence is cleaved, and then one or several mutated bases are inserted when the cleavage site is repaired using the introduced DNA fragment as a template. The several bases are usually 2 to 6 bases.

The CRISPR/Cas9 system consists of three factors: CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein. The Cas9 protein derived from Streptococcus pyogenes recognizes the three bases NGG (wherein N is G, A, T, or C) downstream of the target genome sequence as a PAM sequence (Proto-spacer Adjacent Motif) and cleaves the sequence upstream of the PAM sequence. A common method in this technical field is to introduce a DNA double-strand break at a targeted site on the genome DNA by expressing a guide RNA (gRNA) in which a tracrRNA is linked to the 3' end of a crRNA complementary to the target gene sequence, and Cas9. In this specification, the guide RNA is designed to suppress or reduce the expression of the LhHFS gene, or to knock out the LhHFS gene. In other words, the guide RNA is designed to express a protein in which an amino acid sequence is partially deleted, substituted, or added to the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

The ZFN (zinc finger nuclease) system uses an artificial restriction enzyme consisting of a zinc finger domain (ZF) that recognizes and binds to target DNA, and a FokI domain that cleaves DNA. In this system, the zinc finger domain recognizes a specific gene sequence on the genome, and the FokI domain cleaves the DNA in this region, resulting in a double-stranded break in the DNA.

The TALEN (Transcription Activator-Like Effector Nucleases) system uses an artificial nuclease that has the TAL effector (TALE), a transcription factor from Xanthomonas bacteria, as its DNA-binding domain. The mechanism of action of genome editing by TALEN is similar to that of ZFN, in that it recognizes and binds to the target DNA, and the FokI domain cleaves the DNA in this region, resulting in double-stranded breaks in the DNA. The DNA-binding domain of TALE has a repeat structure with 34 amino acids per unit (module), and it is known that one module recognizes one base. The 12th and 13th amino acid residues in the module are called repeat variable residues (RVD), and the RVD contributes to base binding specificity and stabilization.

Methods for introducing mutations into target genes by genome editing include, for example, a transformation method using Agrobacterium and a method for directly introducing a genome editing factor into protoplasts. The transformation method using Agrobacterium is a method for introducing a genome editing factor into a plant cell by infecting the plant cell with Agrobacterium bacteria containing a vector that expresses a genome editing factor. That is, plant cells or plant tissues isolated from a target plant (e.g., a lily family plant) are incubated with Agrobacterium bacteria containing a vector that expresses a genome editing factor, and the genome editing factor is introduced into the plant cell. Then, cell culture or tissue culture is performed under appropriate culture conditions to generate a genome-edited callus. Then, a genome-edited plant body is regenerated by inducing differentiation of the genome-edited callus, thereby obtaining a plant body whose entire genome is edited. The method for directly introducing a genome editing factor into a protoplast is a method for directly introducing a genome editing factor into a plant cell without using Agrobacterium and a vector. That is, protoplasts are prepared from plant cells derived from the target plant (e.g., a lily family plant), and genome editing factors are directly introduced into the protoplasts using the PEG method, particle gun method, or the like. The protoplasts are then incubated under appropriate culture conditions to obtain a plant body with an edited genome.

Methods for introducing mutations into genomes using mutants include, for example, selecting mutants with mutations in the gene from among natural mutants, and mutants induced by UV irradiation, radiation such as gamma rays, and chemicals such as ethyl methanesulfonate (EMS) using PCR, TILLING (Targeting Induced Local Lesions in Genomes) method, HRM (High Resolution Melting) method, etc., to obtain mutants with mutations in the gene.

Antisense activity is usually achieved by a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the gene of interest. Such a nucleic acid sequence may be a nucleic acid sequence of at least 9 consecutive nucleotides, more preferably 10 consecutive nucleotides, even more preferably 11 consecutive nucleotides, 12 consecutive nucleotides, 13 consecutive nucleotides, 14 consecutive nucleotides, 15 consecutive nucleotides, 20 consecutive nucleotides, 25 consecutive nucleotides, 30 consecutive nucleotides, 40 consecutive nucleotides, or 50 consecutive nucleotides. Such nucleic acid sequences include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, even more preferably 90% homologous, or 95% homologous to the above-mentioned sequences. Such antisense activity is preferably complementary to the 5'-terminal sequence of the nucleic acid sequence of the gene of interest. Such antisense nucleic acid sequences also include those having one or more nucleotide substitutions, additions and/or deletions relative to the above sequences. Therefore, as used herein, "antisense activity" includes, but is not limited to, a reduction in the expression level of a gene.

General antisense technology is described in textbooks (Murray, JAH eds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). Further research revealed a phenomenon called RNA interference (RNAi), which led to the development of antisense technology. RNAi is a phenomenon in which, when a short double-stranded RNA (about 20 bases) with a sequence homologous to a target gene is introduced into a cell, the mRNA of the target gene that is homologous to the RNA sequence is specifically degraded, resulting in a decrease in the expression level. This phenomenon, which was initially discovered in nematodes, has come to be known to be a universal phenomenon in living organisms, including plants, and it has been elucidated that the molecular-level mechanism by which the expression of a target gene is suppressed by antisense technology goes through a process similar to that of RNAi. Conventionally, a DNA sequence complementary to the nucleotide sequence of the target gene is linked to an appropriate promoter, and an expression vector that expresses an artificial mRNA under its control is constructed and introduced into a cell. In recent findings, expression vectors designed to construct double-stranded RNA within cells are used. The basic structure is created by linking one type of DNA sequence complementary to a certain target gene under a promoter, and then linking another identical one in the opposite direction. In the single-stranded mRNA transcribed from this construct gene, the one type of nucleotide sequence portion linked in the opposite direction is complementary, so it pairs up to form a double-stranded RNA state with a hairpin-like secondary structure, which causes mRNA degradation of the target gene according to the mechanism of RNAi. In plants, there have been reports of its use in Arabidopsis thaliana (Smith, NA et al., Nature 407.319-320, 2000). In addition, a general review of RNAi has been published (Morita and Yoshida, Proteins, Nucleic Acids, and Enzymes 47, 1939-1945, 2002). The contents of these documents are incorporated herein in their entirety by reference.

As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, which is generally known in the art and is a biological process that inhibits or downregulates gene expression in cells, mediated by factors that cause RNAi. For example, it refers to the phenomenon in which homologous mRNA is specifically degraded and synthesis of gene products is suppressed by introducing factors that cause RNAi, such as double-stranded RNA (also called dsRNA), into cells, and the technology used therefor. In this specification, "RNAi" may also be used synonymously with "factors that cause RNAi," "factors that cause RNAi," "RNAi factors," and the like, in some cases. For RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al. , 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbasiretal. , 2001, Nature, 411, 494-498; and Kreutzer et al., WO 00/44895; Zernicka-Goetz et al., WO 01/36646; Fire, WO 99/32619; Plaetinck et al., WO 00/01846; Mello and Fire, WO 01/29058; Deschamps-Depaillette, WO 99/07409 and Li et al., WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al. , 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al. , 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al. , 2002, RNA, 8, 842-850; Reinhart et al. , 2002, gene & Dev. , 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831.) As used herein, the term RNAi is understood to be synonymous with other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, epigenetics, and the like. As used herein, an "RNAi agent" may be anything that causes "RNAi."

As used herein, factors that induce RNAi include, but are not limited to, RNA or variants thereof that contain a double-stranded portion of at least 10 nucleotides in length, including a sequence that has at least about 70% homology to a portion of the nucleic acid sequence of a target gene or a sequence that hybridizes under stringent conditions. Here, the factor preferably contains a 3' overhanging end, and more preferably, the 3' overhanging end may be DNA that is 2 nucleotides or more in length (e.g., DNA that is 2 to 4 nucleotides in length).

Alternatively, the RNAi factors used in the present disclosure may include, but are not limited to, pairs of short inverted complementary sequences (e.g., 15 bp or more, e.g., 24 bp, etc.).

Without being bound by theory, one possible mechanism by which RNAi works is that when a molecule that induces RNAi, such as dsRNA, is introduced into a cell, in the case of a relatively long RNA (e.g., 40 base pairs or more), an RNaseIII-like nuclease called Dicer, which has a helicase domain, cuts out the molecule from the 3' end in units of about 20 base pairs in the presence of ATP, generating short dsRNA (also called siRNA). In this specification, "siRNA" is an abbreviation for short interfering RNA, and refers to short double-stranded RNA of 10 base pairs or more that is artificially chemically synthesized or biochemically synthesized, or synthesized in a living organism, or that is generated by decomposing double-stranded RNA of about 40 bases or more in the body, and usually has a 5'-phosphate, 3'-OH structure, with the 3' end overhanging by about 2 bases. A specific protein binds to this siRNA to form a RISC (RNA-induced-silencing-complex). This complex recognizes and binds to an mRNA having the same sequence as the siRNA, and cleaves the mRNA at the center of the siRNA by RNaseIII-like enzyme activity. It is preferable that the sequence of the siRNA and the sequence of the mRNA to be cleaved as a target are 100% identical. However, for base mutations at positions away from the center of the siRNA, the cleavage activity by RNAi is not completely eliminated, but partial activity remains. On the other hand, the effect of base mutations at the center of the siRNA is large, and the cleavage activity of the mRNA by RNAi is extremely reduced. By utilizing such properties, for mRNAs having mutations, siRNAs with the mutations at the center are synthesized and introduced into cells to specifically degrade only the mRNAs containing the mutations. Therefore, in the present disclosure, siRNA itself can be used as a factor that induces RNAi, and a factor that produces siRNA (e.g., typically dsRNA of about 40 bases or more) can be used as such a factor.

Also, without wishing to be bound by theory, it is contemplated that, apart from the above-mentioned pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP), synthesizing dsRNA, which then serves as a substrate for Dicer again, generating new siRNA and amplifying the effect. Therefore, in the present disclosure, siRNA itself and factors that generate siRNA are also useful. In fact, in insects, for example, 35 dsRNA molecules almost completely degrade 1,000 or more copies of intracellular mRNA, and therefore it is understood that siRNA itself and factors that generate siRNA are useful.

In the present disclosure, double-stranded RNA having a length of about 20 bases (e.g., typically about 21 to 23 bases) or less, referred to as siRNA, can be used. Such siRNA can be used in the treatment, prevention, prognosis, etc. of diseases, as it suppresses gene expression by expressing it in cells and suppresses the expression of pathogenic genes targeted by the siRNA. The siRNA used in the present disclosure may be in any form, as long as it can induce RNAi.

In another embodiment, the factor that causes RNAi of the present disclosure may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3' end. As used herein, "shRNA" refers to a molecule of about 20 base pairs or more that is a single-stranded RNA that contains a partially palindromic base sequence, thereby forming a double-stranded structure within the molecule and forming a hairpin-like structure. Such shRNA is artificially chemically synthesized. Alternatively, such shRNA can be generated by synthesizing RNA in vitro using T7 RNA polymerase from a hairpin-structured DNA in which the DNA sequences of the sense strand and the antisense strand are linked in reverse. Without wishing to be bound by theory, it should be understood that such shRNA, after being introduced into a cell, is decomposed to a length of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) in the cell, and causes RNAi in the same way as siRNA, and has the treatment effect of the present disclosure. It should be understood that such effects are exerted in a wide range of organisms, such as insects, plants, and animals (including mammals). Thus, shRNA can be used as an active ingredient of the present disclosure, since it induces RNAi in the same way as siRNA. shRNA may also preferably have a 3' overhang. The length of the double-stranded portion is not particularly limited, but may be preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3' overhang may preferably be DNA, more preferably at least 2 nucleotides in length, and even more preferably 2 to 4 nucleotides in length. The factor that induces RNAi used in the present disclosure may be either artificially synthesized (e.g., chemically or biochemically) or naturally occurring, and there is no essential difference in the effect of the present disclosure between the two. For chemically synthesized factors, it is preferable to purify them by liquid chromatography or the like.

The factor that induces RNAi used in the present disclosure can also be synthesized in vitro. In this synthesis system, antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and a T7 promoter. When these are annealed in vitro and then introduced into a cell, RNAi is induced through the mechanism described above, and the effect of the present disclosure is achieved. Here, such RNA can be introduced into a cell by any suitable method, such as the calcium phosphate method. The factor that induces RNAi of the present disclosure also includes factors such as single strands that can hybridize with mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the present disclosure.

In this specification, the vector is one that can be replicated and isolated/purified in common bacteria (typically E. coli strains derived from E. coli K12) used in genetic experiments. This is necessary to construct the target gene to be introduced into plants. Specifically, for example, there are commercially available plasmids such as E. coli pBR322 plasmid, pUC18, pUC19, pBluescript, and pGEM-T. When transforming plant cells by directly introducing gene fragments into them using electroporation, polyethylene glycol, or particle gun methods, the gene to be introduced can be constructed using such commercially available common plasmids. In addition, as a special example of a vector, when transforming plant cells using a gene introduction method mediated by Agrobacterium, it is necessary to use a plasmid called a "binary vector" that has nucleotide sequences corresponding to the replication origins of both E. coli and Agrobacterium, and the border sequences (left border and right border) derived from T-DNA that indicate the border regions that can be introduced into plants. For example, pBI101 (commercially available from Clontech), pBIN (Bevan, N., Nucleic Acid Research 12, 8711-8721, 1984), pBINPlus (van Engelen, FA et al., Transformegenic Research 4, 288-290, 1995), pTN or pTH (Fukuoka H et al., Plant Cell Reports 19, 2000), pPZP (Hajdukiewicz P et al., Plant Molecular Biology 25, 989-994, 1994) and the like are included, but are not limited thereto. Another example of a vector that can be used in plants is the tobacco mosaic virus vector, but because this type of vector does not introduce the target gene into the plant chromosome, its use is limited to cases where it is not necessary to propagate the plant with the introduced gene via seeds, but it can be used in the present disclosure.

In the present specification, the technique for introducing a nucleic acid molecule into a cell may be any technique, such as transformation, transduction, transfection, etc. Such techniques for introducing a nucleic acid molecule are well known and commonly used in the art, and are described, for example, in Ausubel F. A. et al. (eds.) (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. and its 3rd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Special Edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997. Gene introduction can be confirmed using the methods described herein, such as Northern blot and Western blot analysis, or other commonly known techniques.

The vector can be introduced by any of the above-mentioned methods for introducing DNA into cells, including transfection, transduction, transformation (e.g., calcium phosphate method, liposome method, DEAE-dextran method, electroporation method, particle gun (gene gun) method, etc.), lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)], and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

As used herein, the term "gene transfer reagent" refers to a reagent used in a gene transfer method to promote transfer efficiency. Examples of such gene transfer reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine-based reagents, polyimine-based reagents, calcium phosphate, and the like. Specific examples of reagents used in transfection include those commercially available from various sources, such as Effectene Transfection Reagent (cat. no. 301425, Qiagen, CA), TransFast™ Transfection Reagent (E2431, Promega, WI), Tfx™-20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301305, Qiagen, CA), and others. Examples of such reagents include, but are not limited to, LipofectAMINE 2000 Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reagent (11668-019, Invitrogen Corporation, CA), JetPEI (x4) conc. (101-30, Polyplus-transfection, France) and ExGen 500 (R0511, Fermentas Inc., MD).

When the present disclosure is used in plants, methods well known to those skilled in the art, such as the Agrobacterium-mediated method and the method of directly introducing the vector into a plant cell, can be used to introduce the plant expression vector into a plant cell. For example, the method of Nagel et al. (Nagel et al. (1990), Microbiol. Lett., 67, 325) can be used as the Agrobacterium-mediated method. This method involves first transforming Agrobacterium with an expression vector suitable for plants, for example, by electroporation, and then introducing the transformed Agrobacterium into a plant cell using the method described in Gelvin et al. (Gelvin et al. (ed.) (1994), Plant Molecular Biology Manual (Kluwer Academic Press Publishers)). Methods for directly introducing a plant expression vector into a cell include electroporation (see Shimamoto et al. (1989), Nature, 338:274-276; and Rhodes et al. (1989), Science, 240:204-207), particle gun method (see Christou et al. (1991), Bio/Technology 9:957-962), and polyethylene glycol (PEG) method (see Datta et al. (1990), Bio/Technology 8:736-740). These methods are well known in the art, and a method suitable for the plant to be transformed can be appropriately selected by those skilled in the art.

Cells into which a plant expression vector has been introduced are first selected for drug resistance, such as hygromycin resistance or kanamycin resistance. They can then be redifferentiated into plant tissues, plant organs and/or plants by methods well known in the art. Furthermore, seeds can be obtained from the plants. Expression of the introduced gene can be detected by Northern blotting or PCR. If necessary, expression of the protein that is the gene product can be confirmed, for example, by Western blotting.

The present disclosure has been shown to be particularly useful in plants, but can also be used in other organisms. The molecular biology techniques used in the present disclosure are well known and commonly used in the art, and are described, for example, in Ausubel F. A. et al. (eds.) (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. and its 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Special Edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, and the like.

Physical transformation methods include the polyethylene glycol (PEG) method, electroporation, microinjection, and particle gun methods. These methods are highly useful because they can be applied to both monocotyledonous and dicotyledonous plants. However, the polyethylene glycol and electroporation methods require the use of protoplasts because the cell wall is an obstacle, and the frequency of incorporation of the introduced gene into the chromosomal DNA of the plant cell is low. In addition, the microinjection method using callus or tissue without using protoplasts has many difficulties regarding the needle thickness and tissue fixation. The particle gun method using tissue also has problems such as the appearance of mutations in the form of chimeras. In addition, in these physical methods, the introduced foreign gene is generally easily incorporated into the nuclear genome as multiple copies of the gene in an incomplete state. It is known that when multiple copies of a foreign gene are introduced, the gene is easily inactivated.

On the other hand, methods for introducing isolated genes using living organisms include the Agrobacterium method, the virus vector method, and a method using pollen as a vector. These methods do not use protoplasts but use plant callus, tissue, or plant body to introduce genes, so they have the advantage that they do not require long-term cultivation and are less susceptible to damage such as somaclonal mutation. Of these, there are still few experimental examples of the method using pollen as a vector, and many unknown aspects remain as a plant transformation method. The virus vector method has the advantage that the gene to be introduced spreads throughout the entire plant body infected with the virus, but has problems in that it is only amplified and expressed in each cell, and there is no guarantee that it will be passed on to the next generation, and long DNA fragments cannot be introduced. The Agrobacterium method has many advantages, such as the ability to introduce DNA of about 20 kbp or more into chromosomes without major rearrangements, the number of copies of the introduced gene is small (only a few copies), and high reproducibility. Since Agrobacterium is outside the host range of monocotyledonous plants such as grasses, the introduction of foreign genes into grasses has traditionally been carried out by physical methods as mentioned above. However, in recent years, the Agrobacterium method has come to be applied to monocotyledonous plants such as rice, for which culture systems have been established, and the Agrobacterium method is now preferred.

When introducing foreign genes using the Agrobacterium method, when low molecular weight phenolic compounds such as acetosyringone synthesized by plants act on the Vir region of the Ti plasmid, the T-DNA region is excised from the Ti plasmid and incorporated into the nuclear chromosomal DNA of the plant cell through several processes. In dicotyledonous plants, the plants themselves have the mechanism for synthesizing such phenolic compounds, so foreign genes can be easily introduced using the leaf disc method, etc., and the reproducibility is high. In contrast, in monocotyledonous plants, the plants themselves do not synthesize such phenolic compounds, so it has been difficult to create transformed plants using Agrobacterium. However, it is now possible to introduce foreign genes into monocotyledonous plants by adding acetosyringone during Agrobacterium infection.

In the present disclosure, in a transformant, the nucleic acid molecule of interest (transgene) may or may not be introduced into a chromosome. Preferably, the nucleic acid molecule of interest (transgene) is introduced into a chromosome, and more preferably, into both of two chromosomes.

(plant)
Preferred plants are not limited to ornamental plants for viewing flowers, but also include any plant for which it is useful to prevent flower senescence, such as crops, trees, lawns, weeds, etc. Unless otherwise specified, plants include any plant body, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, bulbs, leaves, stems, flowers (or parts of organs or tissues that constitute flowers, such as petals or similar organs (e.g., perianth)), pollen, scales, shoots, and bulbils. Plants may also be provided as a combination of plant tissues or plant organs, such as cut flowers or potted flowers (combinations of flowers, stems, leaves, and/or roots). Examples of plant cells include callus and suspension culture cells.

As a method for genome editing in plant cells, any of the methods described in detail elsewhere in this specification can be used, for example, Yan, R.; Wang, Z.; Ren, Y.; Li, H.; Liu, N.; Sun, H. Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven. Int. J. Mol. Sci. 2019, 20, 2920. and the method described in JP 2020-130054 A are examples of such methods.

As a method for introducing a recombinant vector into a plant cell, any method for introducing DNA into a plant cell can be used, as detailed elsewhere in this specification. Examples include Agrobacterium (JP Patent Publication No. 59-140885, JP Patent Publication No. 60-70080, WO94/00977), electroporation (JP Patent Publication No. 60-251887), and a method using a particle gun (gene gun) (Patent No. 2606856, Patent No. 2517813).

In this specification, plants can be cultivated by any method known in the art. Methods for cultivating plants are described in "How to Cultivate Yuri" (edited by Imanishi Hideo, Nobunkyo, 2006) and elsewhere.

For the culture, dedifferentiation, differentiation and regeneration of plant cells, plant tissues and plants, techniques and media known in the art are used. Such media include, but are not limited to, Murashige-Skoog (MS) medium, GaMborg B5 (B) medium, White medium, Nitsch & Nitsch (Nitsch) medium and the like. These media are usually used with the addition of appropriate amounts of plant growth regulators (plant hormones) and the like.

To create a plant cell, plant tissue, plant organ, or plant body or part thereof in which the gene for LhHFS or its homologous gene is mutated or deleted in part or in whole, the whole plant body or part thereof may be mutated by exposing it to a conventional mutagenic factor. Alternatively, a part of the plant body may be dedifferentiated to form callus or cells, and the callus or cells may be modified by genetic engineering or the like to obtain callus or cells containing the factors of the present disclosure or modified with the factors of the present disclosure.

As used herein, in the case of plants, "redifferentiation" refers to the phenomenon in which undifferentiated cells differentiate into more differentiated entities such as functional cells, tissues, or entire organisms. For example, callus can be redifferentiated to form tissue fragments such as cells (leaves, roots, etc.), and these tissue fragments can be grown to form organs or plant bodies.

Methods for regenerating transformants into plants are well known in the art. Such methods include those described in Rogers et al., Methods in Enzymology 118:627-640 (1986); Tabata et al., Plant Cell Physiol., 28:73-82 (1987); Shaw, Plant Molecular Biology: A practical approach. IRL press (1988); Shimamoto et al., Nature 338:274 (1989); Maliga et al. , Methods in Plant Molecular Biology: A laboratory course. Cold Spring Harbor Laboratory Press (1995), but are not limited thereto. Therefore, a person skilled in the art can use the above-mentioned well-known method appropriately depending on the desired transgenic plant to regenerate it. The transgenic plant thus obtained has the desired gene introduced therein, and the introduction of such a gene can be confirmed using the methods described herein, such as Northern blot and Western blot analysis, or other well-known and commonly used techniques.

Preferred Embodiments
The following is a description of a preferred embodiment of the present disclosure. It is understood that the following embodiments are provided for a better understanding of the present disclosure, and the scope of the present invention should not be limited to the following description. Therefore, it is clear that a person skilled in the art can appropriately modify the present invention in accordance with the description in this specification.

LhHFS Gene and Protein
In a representative aspect of the present disclosure, the present disclosure provides a newly discovered LhHFS gene and an LhHFS protein encoded by this gene. LhHFS is one of the transcription factor family genes whose expression level increases during flower senescence, but as a result of producing a transformant (LNR18) in which the expression of LhHFS is suppressed, it was observed in a lily plant (Oriental Hybrid system) that the period until the flower turns brown is extended (see Examples). These results show that LhHFS is a transcription factor family gene that controls (promotes) flower senescence, and that flower senescence can be delayed by suppressing the expression of LhHFS. In Oriental Hybrid systems, the sequence homology of the LhHFS gene between the Tiara variety used in the Examples and other varieties (Casablanca variety and Acapulco variety) is very high at 99% or more, so it is understood that the suppression of senescence by the LhHFS inhibitor is not specific to the variety. In addition, it is understood that the ornamental period can be extended in other lily plants by using LhHFS inhibitors such as genome editing, gene recombination, or inhibitors targeting LhHFS orthologues. In particular, since the homologous gene (having the base sequence described in SEQ ID NO: 4) of the LhHFS gene in tulips (Liliaceae, Tulipa genus) has about 71% sequence identity with the nucleic acid sequence (SEQ ID NO: 1) encoding lily LhHFS, it is believed that the aging of flowers can be delayed by suppressing the expression of a sequence variant having at least about 70% sequence identity. LhHFS proteins have in common at least a region expected to bind to conserved DNA in lily plants. In particular, without wishing to be bound by theory, in liliaceae plants, programmed cell death occurs during flower senescence, and the expression of a common group of genes is induced (see, for example, van Doorn WG, Woltering EJ. Physiology and molecular biology of petal senescence. Journal of Experimental Botany 59:453-80, 2008), so it is understood that a similar control mechanism exists, and that inhibition of the activity of LhHFS orthologs is similarly effective in plants other than lilies. It is understood that the present disclosure exerts similar effects in plants other than those in the examples. The factor of the present disclosure may be any factor that can temporarily or permanently suppress, reduce or eliminate the function of LhHFS, and includes, but is not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), factors for genome editing, polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof, and in some cases may be other elements other than substances (e.g., mutagenic factors such as energy such as light, radiation, heat, and electricity).

The present disclosure is effective in liliaceae plants (e.g., plants of the genus Lilium and tulipa) where the aging effect progresses due to the browning of flowers several days after flowering. Since aging in liliaceae plants (e.g., plants of the genus Lilium and tulipa) is different from the wilting or inrolling of flowers commonly seen in other plants, the aging inhibition effect of the technology disclosed herein is significantly useful. For example, it is also noteworthy that while lilies and tulips are composed of three inner perianths and three outer perianths, the flowers of representative plants other than liliaceae plants are not similarly composed, and are composed of, for example, petals or a corolla in which the petals are fused together, and it is noteworthy that aging can be inhibited even with a structure of three inner perianths and three outer perianths. Furthermore, the effect in monocotyledonous plants is also considered to be noteworthy.

Representative nucleic acid sequences of the LhHFS gene include the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2. Thus, in one aspect, the present disclosure relates to a nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2; or (B) a sequence variant, wherein the (B) sequence variant comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 4 pieces, 25 pieces, 26 pieces, 27 pieces, 28 pieces, 29 pieces, 30 pieces, 31 pieces, 32 pieces, 33 pieces, 34 pieces, 35 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, 40 pieces, 41 pieces, 42 pieces, 43 pieces, 44 pieces, 45 pieces, 46 pieces, 47 pieces, 48 pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100) substitutions, additions and/or deletions relative to the nucleic acid sequence of (A); (B-2) a sequence variant having at least about 70%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about The nucleic acid molecule is provided with a sequence variant having 98% or at least about 99% sequence identity, (B-3) a sequence variant that hybridizes under stringent conditions to the nucleic acid sequence of (A), (B-4) a sequence variant that is an allelic variant of (A), or (B-5) a sequence variant that is a fragment of (A) or (B-1) to (B-4), and the protein encoded by the sequence variant has the biological function of the protein encoded by (A). In a more specific aspect, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 1. In an even more specific aspect, the nucleic acid molecule consists of the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

In one aspect, the present disclosure provides a polypeptide comprising: (a) an amino acid sequence as set forth in SEQ ID NO:2 or a fragment thereof; (b) an amino acid sequence as set forth in SEQ ID NO:2, wherein the polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces The present invention provides a polypeptide comprising: a polypeptide having at least one amino acid mutation selected from the group consisting of substitution, addition and/or deletion, and having the same biological activity as the polypeptide of (a); (c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence shown in SEQ ID NO: 1; (d) a polypeptide encoded by a homologous gene of the amino acid sequence shown in SEQ ID NO: 2; or (e) a polypeptide having an amino acid sequence that is at least about 70%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to any one of the polypeptides of (a) to (d), and having the same biological activity as the polypeptide of (a). In a more specific aspect, the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID NO: 2. In a more specific aspect, the amino acid sequence of the polypeptide consists of the sequence shown in SEQ ID NO:2.

The present disclosure understands that suppression of an LhHFS homologous gene is effective in suppressing flower senescence not only in plants of the genus Liliaceae, but also in any plant having an LhHFS homologous gene, particularly in plants of the Liliaceae family in general. Without wishing to be bound by theory, this is because it is believed that the functions of LhHFS homologous genes are substantially similar at least in plants of the Liliaceae family as a whole, and because, although the LhHFS gene was found in this disclosure, the analysis in this disclosure has revealed that this LhHFS gene is commonly found in plants of the Liliaceae family in general.

<Inhibitory factors for LhHFS>
In one aspect, the present disclosure provides an agent that suppresses the function of the nucleic acid sequence or amino acid sequence described in <LhHFS gene and protein>, or a modified sequence thereof. The agent may be a protein, a polypeptide, an oligopeptide, a peptide, a polynucleotide, an oligonucleotide, a nucleotide, a nucleic acid including RNA or DNA, a polysaccharide, an oligosaccharide, a lipid, a hormone, a ligand, a signaling substance, a synthetic chemical, radiation, or a combination or complex agent thereof. In a more specific aspect, the agent is selected from the group consisting of an antisense molecule, an RNAi agent, an agent for genome editing, and a mutagenesis agent.

In one aspect, the present disclosure provides a factor that suppresses LhHFS or a homologous gene thereof. The homologous gene is (i) a sequence variant of the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, the sequence variant being (B-1) a sequence variant having one or more substitutions, additions and/or deletions in the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, (B-2) a sequence variant having at least about 70% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, (B-3) a sequence variant that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, (B-4) a sequence variant that is an allelic variant of the nucleic acid sequence shown in SEQ ID NO: 1 or the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, or (B-5) a nucleic acid sequence that encodes the nucleic acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2, or a sequence variant that is a fragment of (B-1) to (B-4), and the tag encoded by the sequence variant is The protein may be a sequence variant having the biological function of a protein encoded by a nucleic acid sequence shown in SEQ ID NO:1 or a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2, or (ii) a polypeptide comprising: (a) the amino acid sequence shown in SEQ ID NO:2 or a fragment thereof; (b) a polypeptide in which one or more amino acids have been mutated in the amino acid sequence shown in SEQ ID NO:2, the mutation including substitutions, additions and/or deletions, and which has the same biological activity as the polypeptide of (a); (c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence shown in SEQ ID NO:1; (d) a polypeptide encoded by a gene homologous to the gene encoding the amino acid sequence shown in SEQ ID NO:2; or (e) a polypeptide having an amino acid sequence that is at least about 70% identical to any one of the polypeptides (a) to (d) and which has the same biological activity as the polypeptide of (a). Suppression of LhHFS is achieved by, for example, a method selected from the group consisting of a reduction in gene expression level, a reduction in gene copy number, a reduction in gene amplification, a reduction in RNA activity level, a reduction in mRNA abundance, a reduction in mRNA synthesis rate, a reduction in mRNA stability, a reduction in protein activity level, a reduction in protein synthesis, a reduction in protein abundance, a reduction in protein stability, a reduction in protein enzyme activity, or a combination thereof. The factor that suppresses LhHFS may be any substance that partially or completely suppresses the biological function of the LhHFS gene or protein in a plant body. The factor may be a protein, a polypeptide, an oligopeptide, a peptide, a polynucleotide, an oligonucleotide, a nucleotide, a nucleic acid including RNA or DNA, a polysaccharide, an oligosaccharide, a lipid, a hormone, a ligand, a signaling substance, a synthetic chemical, radiation, or a combination or complex factor thereof. In a more specific aspect, the factor is selected from the group consisting of an antisense molecule, an RNAi factor, a factor for genome editing, and a mutagenic factor. In this specification, it is demonstrated that an LhHFS expression suppression line was created using RNAi factors and genome editing, delaying flower senescence (see Examples).

In one embodiment, the factor is in the form of a nucleic acid. Examples of inhibitors in the form of a nucleic acid include aptamers, ribozymes, antisense oligonucleotides, and RNAi factors such as siRNA. In other embodiments, factors in the form of a protein may also be used. Examples of such protein forms include factors for genome editing such as artificial nucleases, antibodies or antigenic fragments, binding peptides, and peptidomimetics. Alternatively, the factor of the present disclosure may be other molecules such as small molecules.

In one embodiment, the agent is an agent that induces RNA interference of LhHFS in the form of a double-stranded nucleic acid.

In a specific embodiment, the factor of the present disclosure is in a double-stranded form, one strand of which comprises (I) (A) a nucleic acid sequence containing at least about 20 bases, about 50 bases, about 100 bases, about 150 bases, or about 200 bases of the nucleic acid sequence shown in SEQ ID NO: 1 (LhHFS itself) or the sequence encoding SEQ ID NO: 2 (or at least any number between these numbers); or (B) a sequence variant, and the other strand comprises (II) a complementary sequence or an annealing sequence of (I), and the (B) sequence variant is a sequence variant having one or several substitutions, additions, or deletions in (B-1) (A), or a sequence variant having the sequence of (B) (A). or (B-3) a sequence variant that hybridizes under stringent conditions to the sequence of (A); (B-4) a sequence variant that is an allelic variant of (A); or (B-5) a sequence variant that is a fragment of (A) or (B-1) to (B-4), and the protein encoded by the sequence variant has the biological function of the protein encoded by (A).

In a further specific embodiment, the factor of the present disclosure is in a double-stranded form, and one strand (I) does not include the 462-base sequence from bases 22-483 of SEQ ID NO: 1. Without wishing to be bound by theory, the base sequence from bases 22-483 of SEQ ID NO: 1 corresponds to a DNA binding site, and targeting this site may suppress the expression of genes other than the LhHFS gene, so it is desirable to target a region other than this site. In a more specific embodiment, one strand (I) includes a nucleic acid sequence from bases 574-864 of SEQ ID NO: 1. Specific embodiments are not limited thereto, and it is understood that any range of length from 22-483 bases may be used (Wesley et al., Plant Journal 27:581-590). In addition, suitable RNAi factors are described in Wesley et al. The design can be carried out with reference to Plant Journal 27:581-590, etc.

In plant RNAi, it is generally believed that any region of the transcript (mRNA) of a target gene can be used to induce mRNA degradation. Based on the principles of RNAi, it is clear to those skilled in the art that mRNA degradation will occur if the transcribed sequence of the target gene is included (see, for example, Wesley et al. Plant Journal 27:581-590). Any region may be selected, but it is preferable to remove the conserved region from the target sequence, since there is a possibility that the mRNA of a gene having the same sequence as the target gene may be degraded when a sequence of a highly conserved region (a sequence common to multiple genes) is targeted. In the RNAi construct of LhHFS disclosed herein, the relatively highly conserved region was removed as a precaution in the examples. It is known that RNAi can be induced by targeting the 5'UTR and 3'UTR (untranslated regions) instead of the coding region (see, for example, Wesley et al. Plant Journal 27:581-590). In plants, siRNA can also be used, but it is understood that longer RNAi factors can also be used preferably (in plants, longer dsRNA is expressed, but is ultimately processed into siRNA of about 20 bases to induce silencing).

In one aspect, the present disclosure provides a vector comprising a nucleic acid that induces RNA interference of LhHFS. The nucleic acid that induces RNA interference of LhHFS contained herein may be any of the forms exemplified by the factors that inhibit LhHFS of the present disclosure, and it is understood that any form may be used as long as it is a nucleic acid that induces RNA interference of LhHFS.

In another embodiment, the vector of the present disclosure includes a promoter and/or a terminator.

In one aspect, the present disclosure provides a genome editing factor that causes the function and/or suppression of the function of LhHFS. The genome editing factor may be any factor used in techniques such as CRISPR/Cas9, TALEN, ZFN, and meganuclease.

When CRISPR/Cas9 is selected as a genome editing method, a guide RNA having a sequence of 23 bases in total, including NGG (N is any base of ATGC) called a PAM sequence at the 3' end, is used. The sequence of the guide RNA can be designed, for example, by a program such as CRISPR-P. The sequence of the guide RNA used for genome editing is preferably one that targets genome editing in the nucleic acid sequence of bases 1 to 483 of SEQ ID NO: 1, and examples of the sequence of the guide RNA include the sequences shown in the following table.

In one aspect, the present disclosure provides a plant cell containing a factor that suppresses LhHFS of the present disclosure or modified with said factor. In another aspect, a plant tissue containing said factor or modified with said factor or a plant tissue containing said plant cell is provided. In yet another aspect, a plant organ containing said factor or modified with said factor or a plant organ containing said plant cell or said plant tissue is provided. In yet another aspect, a plant body or a part thereof containing said factor or modified with said factor, or a plant body or a part thereof containing said plant cell, said plant tissue, or said plant organ is provided.

In a preferred embodiment, the plant cell of the present disclosure is a plant cell of the lily family. Examples of such plant cells include cells of plants of the genus Lilium, Tulipa, Erythronium, Fritillaria, Amana, Bowiea, Calochortus, Cardiocrinum, Clintonia, and Gagea. , Lloydia, Medeola, Nomocharis, Notholirion, Prosartes, Streptopus, Tricyrtis, and the like, including, but not limited to, lilies, tulips, dogtooth violets, and Japanese black lilies. In a further preferred embodiment, the plant cells of the present disclosure are lily cells.

In one embodiment, the plant or part thereof containing the factor that inhibits LhHFS includes a bulb, a seed, a flower, a stem, a leaf, a pollen, a scale, a kernel, a cell, a tissue, a tissue culture, or a cornel, or a combination of all or part thereof. In a specific embodiment, it may be provided in the form of, for example, a whole flower, or an organ constituting a flower such as petals, a corolla, and a perianth, or a combination of organs comprising a flower (e.g., a cut flower or a potted flower, etc.).

(Method for producing a plant in which LhHFS or its homologous gene is suppressed)
In another aspect, the present disclosure provides a plant cell in which LhHFS is suppressed more than in the natural state. The "natural state" refers to a state in which the expression level of LhHFS at the peak of expression is equivalent to that of the housekeeping gene Elongation factor 1 alpha (EF1alpha). Here, the expression level of LhHFS is low on day 0 after flowering, but reaches its peak on about day 4 to 5 after flowering, and therefore the expression level on about day 4 to 5 after flowering is used.
In such cells, the state in which LhHFS is suppressed more than in the natural state can be achieved by introducing the LhHFS suppressing factor of the present disclosure into the plant cell. When the plant cell is obtained using a factor that suppresses LhHFS, conventionally, transduction is performed using a vector containing a drug resistance gene (e.g., a hygromycin resistance gene), and a plant cell into which a mutation has been inserted can be screened by screening using a drug resistance marker. Alternatively, a plant in which LhHFS is suppressed may be selected by PCR or the like from a group of plants or plant cells, etc., in which a mutation has occurred in LhHFS, from among the plants or plant cells, etc., which have been subjected to a mutation treatment using a mutagenic agent or the like. In this case, for example, in the case of lilies, plants with a lower level of expression or activity than the natural LhHFS disclosed in the present disclosure can be selected. In the case of other plants, selection can be made in the same manner. Alternatively, in addition to searching by a method of introducing a suppressing factor or screening, a method of modifying the sequence of endogenous LhHFS using a method such as zinc finger nuclease or gene targeting to suppress expression may be used. In carrying out the present disclosure, with regard to the application of zinc finger nucleases in plants, Keishi Osakabe, Yuriko Osakabe and Seiichi Toki, Site-directed mutagenesis in Arabidopsis using custom-designed zinc-finger nucleases. Proc. Nat. Acad. Sci. USA (2010) June 29, 107 (26): 12034-12039 can be referred to. Regarding gene targeting, see Molecular breeding of a novel herbicide-tolerant rice by gene targeting. Endo, Masaki; Osakabe, Keishi; Ono, Kazuko; Handa, Hirokazu; Shimizu, Tsutomu; Toki, Seiichi. The Plant Journal vol. 52 issue 1 October 2007. p. 157-166; Rie Terada, Hiroko Urawa, Yoshishige Inagaki, Kazuo Tsugane, and Shigeru Iida: Efficient gene targeting by homologous recombination in rice. Nature Biotechnology 20(10)1030-1034 (2002) can be referred to.

The plant cells, tissues, organs, or plants obtained by screening can be confirmed to have suppressed LhHFS compared to the natural state by a method selected from the group consisting of, for example, a decrease in gene expression level, a decrease in gene copy number, a decrease in gene amplification, a decrease in RNA activity level, a decrease in mRNA abundance, a decrease in mRNA synthesis rate, a decrease in mRNA stability, a decrease in protein activity level, a decrease in protein synthesis, a decrease in protein abundance, a decrease in protein stability, a decrease in protein enzyme activity, or a combination thereof. The decrease in gene expression level can be confirmed typically by a method such as real-time PCR (RT-PCR). The decrease in protein expression level can be confirmed typically by a method such as Western Blotting. In addition, in the case of LhHFS, the decrease in protein enzyme activity can be measured by the expression level of downstream genes, a reporter assay, or the like.

Screening may be performed in any state, such as in cells, tissues, organs, or plants. Preferably, screening for drug resistance is performed in callus. The presence or absence of gene introduction and the presence or absence of mutation introduction by genome editing are analyzed by PCR or sequencing using DNA extracted from adventitious buds (shoots) induced from the callus. The reduction in LhHFS gene expression and protein amount is analyzed in the perianth.

Plant cells having the desired traits of this disclosure can be redifferentiated into tissues, organs, or whole plants by methods known in the art.

Once a plant body having the desired traits of this disclosure is obtained as a transformant, the transformant may be used as a pollen parent or seed parent to create a new, separate variety having the desired traits of this disclosure by conventional crossing methods.

In a preferred embodiment, the plant cell of the present disclosure is a plant cell of the lily family. Examples of such plant cells include cells of plants of the genus Lilium, Tulipa, Erythronium, Fritillaria, Amana, Bowiea, Calochortus, Cardiocrinum, Clintonia, and Gagea. , Lloydia, Medeola, Nomocharis, Notholirion, Prosartes, Streptopus, Tricyrtis, and the like, including, but not limited to, lilies, tulips, dogtooth violets, and Japanese black lilies. In a further preferred embodiment, the plant cells of the present disclosure are lily cells.

In one embodiment, the suppression of flower aging in the present disclosure may be suppression of aging of some or all of the elements constituting a flower. The elements constituting a flower include the following: petals, corolla, perianth, stamens, pistil, sepals, receptacles, and flower axes. The suppression of flower aging may be suppression of aging of at least one, at least two, at least three, at least four, or more of the elements constituting a flower. In a preferred embodiment, it may be advantageous if the aging of any of the elements constituting a flower, such as the petals, corolla, and perianth, is suppressed. In a preferred embodiment, the suppression of flower aging may be achieved by suppressing the aging of any one of the petals, corolla, and perianth, or may be achieved by suppressing the aging of the entire flower. With regard to the suppression of flower aging, it is not necessary to achieve the suppression of aging of the stamens, pistils, etc. In various embodiments, inhibition of flower senescence typically refers to senescence of the "petals," but may also refer to senescence of the corolla, senescence of the perianth, senescence of the perianth attached to the receptacle, senescence of the entire flower excluding the stamens and pistil, or senescence of the entire flower (i.e., including all the elements that make up the flower, such as the perianth, the rachis which corresponds to the stem, the stamens, the pistils, etc.).

(How to extend the shelf life of flowers)
In another aspect, the present disclosure provides a method for extending the shelf life of flowers in a plant or part thereof. The method for extending the shelf life of flowers includes the steps of: 1) providing a cell (e.g., a plant cell), a plant tissue, or a plant or part thereof that contains or is modified by a factor described in <Suppressing factor of LhHFS>; 2) subjecting the cell, plant tissue, or plant or part thereof to flowering conditions; and 3) subjecting the cell, plant tissue, or plant or part thereof to conditions and periods that allow the plant or part thereof to flowering. The period may be longer than the period that the plant or part thereof would end its shelf life under the conditions that allow the plant or part thereof to flower without modification by the factor. For example, when "Tiara", a variety of the oriental hybrid lineage of lily, is used, the period during which the shelf life ends without modification with the factor is about 5 days, and in this case, the period during which the shelf life ends for the plant or part thereof modified with the factor may be 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, etc. In a preferred embodiment, the plant or part thereof may be the entire flower, or organs constituting the flower such as petals, corolla, and perianth, or a combination of organs including the flower (e.g., cut flowers or potted flowers, etc.).

(screening)
In another aspect, the present disclosure provides a method for screening an agent that delays flower senescence using the nucleic acid molecule (LhHFS or its variant) of the present disclosure or the protein encoded by it. As the screening method, a method known in the art can be applied. This method includes (i) contacting a test substance with LhHFS or a substance, cell, plant, etc. that contains it; and (ii) detecting the expression of LhHFS in the substance, cell, plant, etc. after contacting the test substance. In step (i), the cells, etc. used are those of a Liliaceae plant. The agent that delays flower senescence can be selected from substances or factors that suppress the expression or activity of LhHFS in the test substance. Such substances or factors can be further introduced into a plant or plant cell to confirm whether they can delay flower senescence.

(General Technology)
The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). These are described in PCR Applications: Protocols for Functional Genomics, Academic Press, Special Edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, "PCR Experimental Protocols for Plants," Shujunsha (1997), and Experimental Protocols for Model Plants, Shujunsha (1996), the relevant portions of which (possibly in their entirety) are incorporated herein by reference.

For DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.; M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (I996). These are described in Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth herein.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

Where necessary, the plants used in the following examples were handled in compliance with the standards set by the Japanese government, the National Agriculture and Food Research Organization, and the Niigata Prefectural Agricultural Research Institute. In addition, the reagents used were specifically the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai Tesque, Kanto Chemical, etc.) can also be used.

(Plant materials and growth conditions)
In the following examples, the plant used was "Tiara," a variety of the oriental hybrid lily. "Tiara" was obtained from domestic bulb sales companies such as Yamaki Farm Co., Ltd. or Nakamura Farm Co., Ltd. Bulbs were planted in horticultural soil (adjusted peat moss, BVB) and cultivated in a greenhouse. Flower shelf life was evaluated by cutting flowers immediately after flowering from the plant body, placing them in distilled water, and keeping them indoors (temperature 20°C, PPFD 10 μmol m ^-2 s ^-1 , day length 12 hours). The shelf life was determined to be over when at least half of the perianths had turned brown, or when at least half or more of the perianths were found to have turned brown.

Example 1: Isolation of LhHFS
The present inventors conducted extensive research into the lily genome and discovered a novel gene that may be related to flower senescence. This gene was isolated, and 3'-RACE and 5'-RACE were performed based on the isolated partial sequence to determine the base sequence including the full length of the ORF. RNeasy plant mini kit (Qiagen) was used for RNA extraction, and PrimeScript RT Master Mix (Takara bio) was used for cDNA synthesis. PrimeSTAR HS DNA polymerase (Takara bio) was used for PCR. A cDNA clone containing the full-length ORF was obtained by RT-PCR using a full-length cloning primer set (LhHFS-Full-F9: 5'-TTCCAACTTTCAACCTTCAACTC-3' (SEQ ID NO: 6), LhHFS-Full-R1188: 5'-TTGTCGACATAAATTGATCCAAG-3' (SEQ ID NO: 7)). The obtained cDNA clone was cloned into pGEM-T easy vector (Promega), and the nucleotide sequence was determined and named LhHFS.

(Example 2: Creation of LhHFS expression-suppressing transformant)
Transformants suppressing the expression of LhHFS were produced using the RNAi method. A 291-base pair LhHFS cDNA fragment (574-864 bases) was cloned into pDONR201 (Invitrogen), and then inserted into the plant transformation vector pH7GWIWG(II) (Karimi M, et al. 2002. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193-195.) carrying the hygromycin resistance gene (HPT) using LR ClonaseII Enzyme mix (Invitrogen). The resulting transformation vector pH7-HFS contains the cDNA fragment of LhHFS in the antisense and sense directions downstream of the cauliflower mosaic virus 35S promoter, and the 35S terminator downstream of the cDNA fragment. pH7-HFS was introduced into cultured cells derived from the filaments of the lily cultivar "Tiara" using Agrobacterium EHA101 strain. Specifically, the filaments of the lily cultivar "Tiara" were cultured in a callus induction medium (MS medium containing 4 g L ^-1 gellan gum and 2 mg L ^-1 picloram, and excluding _MgSO4.7H2O ) to induce callus. The callus was then subcultured in an adventitious shoot regeneration medium (medium with the above composition, but with 0.1 mg L ^-1 picloram added, and 1 _mg L ^-1 thidiazuron added) that simultaneously induces proliferation and regeneration, and cultured for 14 days. After inoculation of Agrobacterium EHA101 strain carrying the transformation vector pH7-HFS into the callus, the callus was selected on an adventitious shoot regeneration medium supplemented with 15 mg L ^-1 hygromycin. The regenerated adventitious shoot was transferred to a bulb enlargement medium (MS medium supplemented with 4 g L ^-1 gellan gum and excluding hormones) every 3 weeks to promote bulb formation, and after about 12 months, it was transferred to horticultural soil in a special netted room for acclimatization. The acclimatized bulbs were grown and allowed to flower. Two types of LhHFS expression suppression lines (LNR18) were obtained (LNR18-2 and LNR18-15). The shelf life of the flowering lilies was evaluated by the above method. The presence or absence of the introduced gene was confirmed by PCR amplification of the HPT gene.

(Results and Discussion)
The shelf life of the wild type and the LhHFS expression suppression line (LNR18) is shown in Table 1. In Table 1, the shelf life of the wild type was 5 days, while the shelf life of the two strains of the LhHFS expression suppression line was 8 days. Photographs of the wild type and the LhHFS expression suppression line from flowering to senescence are shown in FIG. 1. In FIG. 1, in the wild type, three or more perianths were partially browned on the 5th day after flowering, whereas in the LhHFS expression suppression line, no browning of the perianth was observed even on the 7th day after flowering, and three or more perianths were partially browned on the 8th day. This result shows that suppression of LhHFS delays the senescence of the perianth compared to the wild type, and extends the shelf life (ornamental period) of the flower by about 1.5 to 2 times.

Example 3: Analysis of gene expression by quantitative real-time RT-PCR
RNA was extracted from the inner perianth of the wild type and LhHFS expression suppression line on the fourth day after flowering using RNeasy plant mini kit (Qiagen). PrimeScript RT Master Mix (Takara bio) was used for cDNA synthesis, and SYBR Premix EX TaqII (Takara bio) was used for PCR. PCR was performed using Thermal Cycler Real Time System (Model TP600, Takara bio). The primers used for amplifying LhHFS in PCR were LhHFS-907Fq (5'-AAGCAGGGGATCAGACAGAG-3' (SEQ ID NO: 8)) and LhHFS-1013Rq (5'-TTGTTGCCACTCCGAGTAT-3' (SEQ ID NO: 9)). The expression level was normalized using the internal control LhEF1alpha and calculated as a relative expression level. The primers used for amplifying LhEF1alpha were LhEF1a-QF2-729 (5'-TGAGGTGTTCTTCTATTCATGTCC-3' (SEQ ID NO: 10)) and LhEF1a-QR2-867 (5'-CCAAATAGAGCAGCAAAGCA-3' (SEQ ID NO: 11)).

(Results and Discussion)
The relative expression levels of the LhHFS gene in the perianth on day 4 after flowering for the wild type and the LhHFS expression-suppressed lines are shown in Table 2 and Figure 2 below. All of the LhHFS expression-suppressed lines (LNR18-2, LNR18-15) showed greater reduction in LhHFS gene expression than the wild type. These results showed that the LhHFS expression-suppressed lines created in this example had LhHFS expression suppressed to more than twice that of the wild type.

(Example 4: Creation of a transformant in which a mutation has been introduced into LhHFS by genome editing)
A genome editing method using CRISPR/Cas9 is used to introduce a mutation into LhHFS to produce a transformant in which LhHFS expression is suppressed or deleted. A gene expression cassette expressing a guide RNA and a Cas9 gene is inserted into a plant transformation vector pIG121-Hm (Ohta et al., Plant Cell Physiol. 31: 805-813 (1990)) having a hygromycin resistance gene (HPT). The resulting transformation vector pIG-HFS contains a gene expression cassette containing a Cas9 gene downstream of a cauliflower mosaic virus 35S promoter and an actin terminator downstream thereof, and a gene expression cassette containing a guide RNA sequence listed in Table 3 downstream of a rice U6 promoter and a guide RNA scaffold sequence and a polyT sequence downstream thereof. pIG-HFS is introduced into cultured cells derived from filaments of the lily cultivar "Tiara" using Agrobacterium EHA101 _strain . Specifically, filaments of the lily cultivar "Tiara" are cultured in a callus induction medium (MS medium containing 4 g L ^-1 gellan gum and 2 mg L ^-1 picloram and excluding _MgSO4.7H2O ) to induce callus. The callus is subcultured in an adventitious shoot regeneration medium (medium with the above composition, but with 0.1 mg L- ¹ picloram added and 1 mg L ^-1 thidiazuron added) that simultaneously induces proliferation and redifferentiation, and cultured for 14 days. After inoculating the Agrobacterium EHA101 strain carrying the transformation vector pIG-HFS into the callus, it is selected in an adventitious shoot regeneration medium containing 15 mg L ^-1 hygromycin. The regenerated adventitious buds are transplanted every three weeks into a bulb enlargement medium (MS medium containing 4 g L ^-1 gellan gum and no hormones) to promote bulb formation, and after approximately 12 months, they are transplanted into horticultural soil in a special screened greenhouse for acclimatization. The acclimatized bulbs are grown and allowed to flower. The presence or absence of the introduced gene is confirmed by PCR amplification of the HPT gene using DNA extracted from the adventitious buds. The presence or absence of the introduction of a mutation is confirmed by amplifying the region containing the target sequence by PCR using DNA extracted from the adventitious buds and sequencing it.

Alternatively, the introduction of mutations by genome editing can be achieved by directly injecting a complex consisting of guide RNA and Cas9 protein into plant cells by particle bombardment without integrating the Cas9 gene and guide RNA expression cassette into the plant genome. For example, Zhen Liang, Kunling Chen, Yi Zhang, Jinxing Liu1, Kangquan Yin, Jin-Long Qiu & Caixia Gao. Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoprotein. An example of such a method is described in Nature protocol 13:413-430. Specifically, a guide RNA and a Cas9 protein complex are introduced into the shoot apical meristem of a lily using particle bombardment. The shoot apical meristem after the introduction treatment by particle bombardment is cultured by a method known in the art to regenerate adventitious buds. DNA is extracted from the regenerated adventitious buds, and the region containing the target sequence is sequenced to confirm the presence or absence of mutation. The adventitious buds in which the introduction of mutations has been confirmed are transplanted every three weeks to a bulb enlargement medium (MS medium containing 4 g L ⁻¹ gellan gum and excluding hormones) to promote the formation of bulbs, and after about 12 months, they are transplanted to horticultural soil in a specific netted room for acclimatization. The acclimatized bulbs are grown and allowed to bloom.

(Example 5: Verification of LhHFS expression in perianth of different lily varieties)
In this example, the expression of LhHFS in the perianth is verified using varieties of lilies other than "Tiara". Primers for amplifying LhHFS are designed based on the LhHFS gene sequence of "Tiara", and LhHFS of the target lily is isolated by performing RT-PCR using RNA extracted from the perianth of the target lily. After determining the base sequence, the expression of LhHFS is analyzed by real-time PCR. Specifically, RNA is extracted from the perianth using RNeasy plant mini kit (Qiagen), and cDNA is synthesized using PrimeScript RT Master Mix (Takara bio). PrimeSTAR HS DNA polymerase (Takara bio) is used for PCR. The obtained cDNA clone is cloned into pGEM-T easy vector (Promega) and the base sequence is determined. For expression analysis, RNA is extracted from the inner perianth of the flower on the fourth day after flowering using RNeasy plant mini kit (Qiagen), and cDNA is synthesized using PrimeScript RT Master Mix (Takara bio). Then, PCR is performed using SYBR Premix EX TaqII (Takara bio). For PCR, Thermal Cycler Real Time System (Model TP600, Takara bio) is used. The expression level of LhHFS is normalized using the internal control LhEF1alpha, and calculated as a relative expression level.

Example 6: Screening of inhibitors
We will isolate proteins that interact with LhHFS protein and develop a system that can monitor the interaction with LhHFS protein in a test tube (96-well plate). Specifically, we will use reporters such as fluorescent proteins to measure the degree of inhibition of the interaction by compounds. In carrying out this work, we can refer to the methods used in cancer research to search for molecular targeted therapeutic drugs. Using the developed monitoring system, we will perform chemical screening using a compound library to find drugs that inhibit the activity of LhHFS protein.

Although the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth in this specification.

The present disclosure may be used in the floriculture field. Flowering plants (e.g., lilies) with extended flower life are commercially valuable. In other lily family plants such as tulips, suppressing the expression of LhHFS orthologs may also delay flower senescence.

SEQ ID NO:1: The nucleic acid sequence corresponding to the translated region of LhHFS.
SEQ ID NO:2: Amino acid sequence of the LhHFS protein.
SEQ ID NO: 3: Full length nucleic acid sequence of LhHFS including untranslated regions.
SEQ ID NO: 4: Partial length nucleic acid sequence of the homologous gene of LhHFS in tulip.
SEQ ID NO: 5: Partial amino acid sequence of the homologous gene of LhHFS in tulip.
SEQ ID NO: 6: Sequence of the primer used for cloning the LhHFS gene.
SEQ ID NO: 7: Sequence of the primer used for cloning the LhHFS gene.
SEQ ID NO: 8: Sequence of the primer used for RT-PCR of the LhHFS gene.
SEQ ID NO: 9: Sequence of the primer used for RT-PCR of the LhHFS gene.
SEQ ID NO: 10: Sequence of a primer used in RT-PCR of the LhEF1alpha gene.
SEQ ID NO: 11: Sequence of a primer used in RT-PCR of the LhEF1alpha gene.
SEQ ID NOs: 12-95: Sequences of guide RNAs used in genome editing.

Claims (5)

(A) a nucleic acid sequence encoding the nucleic acid sequence set forth in SEQ ID NO:1 or the amino acid sequence set forth in SEQ ID NO:2; or (B) a nucleic acid molecule comprising a sequence variant,
The sequence variant of (B) is a sequence variant having at least 90% sequence identity to the nucleic acid sequence of (A), and the protein encoded by the sequence variant has the biological function of the protein encoded by the nucleic acid sequence of (A), the biological function including LhHFS transcription activity, and the LhHFS transcription activity can be detected by RT-PCR using a vacuum processing enzyme gene (VPE gene) or a cysteine gene.
A nucleic acid molecule, which is measured by measuring the expression of a protease gene (SAG12 gene). The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule consists of the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule consists of the nucleic acid sequence shown in SEQ ID NO:1. (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2 and having the same type of biological activity as the biological activity of the polypeptide of (a), wherein the biological activity includes LhHFS transcription activity, and the LhHFS transcription activity is measured by measuring the expression of the vacuum processing enzyme gene (VPE gene) or the cysteine protease gene (SAG12 gene) using the RT-PCR method . The polypeptide of claim 4, wherein the polypeptide consists of the amino acid sequence shown in SEQ ID NO:2.